Il-7-fc-fusion proteins

ABSTRACT

Provided herein are dimeric IL-7-Fc fusion proteins that include Fc domains and one or more IL-7s. Also provided herein are variant IL-7s with modifications to reduce heterogeneity and/or reduced affinity/potency. Such variant IL-7s are useful, for example, in the subject dimeric IL-7-Fc fusion proteins. The dimeric IL-7-Fc fusion proteins can be used for applications where increased IL-7 activity is useful, for example, for increasing the proliferation of lymphocyte populations in mounting an anti-tumor response in a subject in need thereof.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Application No.62/849,684, filed May 17, 2019, which are hereby incorporated byreference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 29, 2020, isnamed 067461-5239-US_SL.txt and is 396,683 bytes in size.

BACKGROUND

In order for the immune system to mount an effective anti-tumorresponse, two things must occur. T cells in the tumor environment mustfirst engage antigenic tumor peptides presented by majorhistocompatibility complexes (MHC) on tumor cells. Next, the T cellsmust be induced by cytokines such as IL-15 and IL-2 to producecostimulatory cytokines such as IFNγ. Recognition of tumor peptidesalone in the absence of cytokine induction leads to T cells becominganergic, thereby leading to tolerance. Accordingly, a very promisingapproach in cancer immunotherapy is cytokine-based treatments. Forexample, IL-2 has been approved for use in patients with metastaticrenal-cell carcinoma and malignant melanoma.

IL-7 is another cytokine that exerts cell signaling through the commongamma chain (γC; CD132) which is shared by IL-15 and IL-2, in additionto a unique IL-7 receptor (IL-7Rα). Recombinant IL-7 is a promisingcytokine-treatment due to its broad effect in activating the immunesystem as IL-7 signaling contributes to survival, proliferation, anddevelopment of naive and memory B and T cells, mature T cells, and NKcells. However, while there have been several clinical trialsinvestigating IL-7 fusions in treatment of cancer, there are currentlyno approved uses of recombinant IL-7 in humans. Thus, there remains aneed for novel IL-7 based compositions for the treatment of cancers.

BRIEF SUMMARY

Provided herein are dimeric IL-7-Fc fusion proteins that include Fcdomains and one or more IL-7s. As discussed herein, such IL-7-Fc fusionproteins exhibit IL-7 biological activity, and long serum half-lives.Due to the long serum half-lives, the fusion proteins advantageously donot require high doses for use in treatments, thereby minimizing anypotential systemic toxicity associated with increased IL-7 levels. Thedimeric IL-7-Fc fusion proteins can be used for applications whereincreased IL-7 activity is useful, for example, for increasing theproliferation of lymphocyte populations in mounting an anti-tumorresponse in a subject in need thereof. Also provided herein are variantIL-7s with modifications to reduce heterogeneity and/or reducedaffinity/potency. Such variant IL-7s are useful, for example, in thesubject IL-7-Fc fusions.

In a first aspect, provided herein is a dimeric Fc fusion protein thatincludes: (a) a first monomer that includes a first IL-7 and a first Fcdomain, where the IL-7 is covalently attached to the first Fc domain;and (b) a second monomer that includes a second IL-7 and a second Fcdomain, wherein the second IL-7 is covalently attached to the second Fcdomain.

In some embodiments, the first and second IL-7s are identical. Incertain embodiments, the first and/or second Fc domains includes one ormore amino acid substitutions, where the set of amino acidsubstitution(s) are one of the following: C219S, C220S, S228P,G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,E233P/L234V/L235A/G236del/S267K, andC220S/E233P/L234V/L235A/G236del/S267K according to EU numbering.

In some embodiments, the first and second Fc domains each includemodifications C220S/E233P/L234V/L235A/G236del/S267K, according to EUnumbering. In an exemplary embodiment, the first and second and/orsecond Fc domains includes a further amino acid substitution selectedfrom: M428L, N434S, and M428L/N434S, according to EU numbering.

In an exemplary embodiment, the first IL-7 is covalently attached to theN-terminus of the first Fc domain and the second IL-7 monomer domain iscovalently attached to the N-terminus of the second Fc domain. Inanother embodiment, the first IL-7 is covalently attached to theC-terminus of the first Fc domain and the second IL-7 monomer domain iscovalently attached to the C-terminus of the second Fc domain.

In some embodiments of the dimeric fusion protein, the first IL-7 isattached to the first Fc domain using a first domain linker and/or thesecond IL-7 is attached to the second Fc domain using a second domainlinker. In some embodiments, the domain linker is selected from any oneof the domain linkers in FIG. 7. In certain embodiments, the first IL-7is directly attached to the first Fc domain and/or the second IL-7 isattached to the second Fc domain.

In one embodiment, the first and second monomer each includes an aminoacid sequence selected from the following: SEQ ID NO:60 (XENP27088); SEQID NO:61 (XENP27089); and SEQ ID NO:66 (XENP27090).

In an exemplary embodiment of the dimeric fusion protein, the first andsecond IL-7 are each a variant IL-7 that includes one or more amino acidsubstitutions, wherein the amino acid substitutions comprise: N70D,N70Q, N70V, T72V, N91D, N91Q, N91A, N116D, N116Q, N116A,N70D/N91D/N116D, N70Q/N91Q/N116Q, N70A/N91A/N116A, Q11E, Q22E, I30H,L35Q, L35N, D48N, N50D, E52Q, M69S, M69Q, D74N, D74E, K81R, K81E, E84Q,I88T, I88R, L128R, L128Q, E137Q, N143D, D74N/E84Q, D74N/K81R, andD74N/K81E.

In an exemplary embodiment, the first and second monomer each comprisesan amino acid sequence selected from the group consisting of: SEQ IDNO:108-136 (XENP28754-28782).

In a second aspect, provided herein is a heterodimeric Fc fusion proteinthat includes: a) a first monomer that includes a first Fc domainwithout an IL-7 (i.e., an “empty Fc domain”); and b) a second monomerthat includes an IL-7 and a second Fc domain, wherein the IL-7 iscovalently attached to the second Fc domain. Further, the first and thesecond Fc domains comprise modifications promoting heterodimerization ofthe first and the second Fc domains.

In some embodiments, the IL-7 is attached to the N-terminus of thesecond Fc domain. In other embodiments, the IL-7 is attached to theC-terminus of the second Fc domain. In an exemplary embodiment, thefirst monomer consists of the first Fc domain.

In certain embodiments, the modifications promoting heterodimerizationof the first and second Fc domains are a set of amino acid substitutionsselected from the group consisting of L368D/K370S and S364K; L368D/K370Sand S364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E andD401K; L368E/K370S and S364K; K370S and S364K/E357Q andT366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C), according to EU numbering.

In some embodiments, the IL-7 is attached to the second Fc domain usinga domain linker. In an exemplary embodiment, the domain linker isselected from any one of the domain linkers in FIG. 7. In otherembodiments, the IL-7 is directly attached to the second Fc domain.

In one embodiment, the first and/or the second Fc domains have anadditional set of amino acid substitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,E233P/L234V/L235A/G236del/S267K, and C219S, C220S, S228P, G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,E233P/L234V/L235A/G236del/S267K, andC220S/E233P/L234V/L235A/G236del/S267K according to EU numbering.

In some embodiments, first and/or second Fc domains further includes oneor more amino acid substitution(s) selected from the following: M428L,N434S, and M428L/N434S, according to EU numbering.

In one embodiment, the heterodimeric fusion protein includes a firstmonomer and a second monomer having the amino acid sequence of the firstmonomer and second monomer, respectively, of any one of theheterodimeric proteins selected from the group consisting of XENP27079(SEQ ID NOs: 62 and 63), XENP27080 (SEQ ID NOs: 64 and 65); andXENP27083 (SEQ ID NOs. 67 and 68).

In one embodiment of the dimeric fusion protein, the IL-7 is a variantIL-7 comprising one or more amino acid substitutions, wherein the aminoacid substitution(s) is one of the following: N70D, N70Q, N70V, T72V,N91D, N91Q, N91A, N116D, N116Q, N116A, N70D/N91D/N116D, N70Q/N91Q/N116Q,N70A/N91A/N116A, Q11E, Q22E, I30H, L35Q, L35N, D48N, N50D, E52Q, M69S,M69Q, D74N, D74E, K81R, K81E, E84Q, I88T, I88R, L128R, L128Q, E137Q,N143D, D74N/E84Q, D74N/K81R, and D74N/K81E as compared to wild typeIL-7.

In some embodiments, the heterodimeric fusion protein includes a firstmonomer and a second monomer having the amino acid sequence of the firstmonomer and second monomer, respectively, of any one of theheterodimeric proteins selected from the group consisting ofXENP29187-XENP29202.

In another aspect, provided herein is a composition that includes avariant human IL-7. The variant human IL-7 includes one or more aminoacid substitutions, wherein the amino acid substitution(s) are one ofthe following: N70D, N70Q, N70V, T72V, N91D, N91Q, N91A, N116D, N116Q,N116A, N70D/N91D/N116D, N70Q/N91Q/N116Q, N70A/N91A/N116A, Q11E, Q22E,130H, L35Q, L35N, D48N, N50D, E52Q, M69S, M69Q, D74N, D74E, K81R, K81E,E84Q, I88T, I88R, L128R, L128Q, E137Q, N143D, D74N/E84Q, D74N/K81R, andD74N/K81E as compared to wild type human IL-7. In some embodiments, thevariant human IL-7 includes an additional 1, 2, 3, 4, 5, 6, 7, 8, 9, or10 amino acid modifications. In certain embodiments, the variant humanIL-7 exhibits reduced binding to IL-7R. In an exemplary embodiment, thevariant human IL-7 exhibits reduced heterogeneity.

In one aspect, provided herein is a pharmaceutical composition thatincludes any one of the dimeric fusion proteins or subject variant IL-7sdescribed herein.

In another aspect provided herein are nucleic acids encoding any of thesubject dimeric fusion proteins (e.g., subject heterodimeric fusionproteins) or subject variant IL-7s described herein, expression vectorsthat include such nucleic acids and host cells that include suchexpression vectors or nucleic acids. Further provided are methods ofmaking the subject dimeric fusion proteins (e.g., subject heterodimericfusion proteins) and subject variant IL-7s described herein.

In another aspect, provided herein is a method of inducing STAT5phosphorylation in a lymphocyte. This method includes a step ofcontacting the lymphocyte with a composition that includes any of thesubject dimeric fusion proteins, subject heterodimeric fusion proteins,or subject variant IL-7s described herein.

In one aspect provided herein is a method of inducing STAT5phosphorylation in a lymphocyte in a subject comprising administering tothe subject a composition that includes any of the subject dimericfusion proteins, subject heterodimeric fusion proteins, or subjectvariant IL-7s described herein.

In some embodiments of the above methods, the lymphocyte is aCD4⁺CD45RA⁺ lymphocyte, a CD4⁺CD45RA⁻ lymphocyte, a CD56⁺ NK cell, or aTreg cell.

In another aspect, provided herein is a method of inducing Ki67expression in a lymphocyte comprising contacting the lymphocyte with acomposition that includes any of the subject dimeric fusion proteins,subject heterodimeric fusion proteins, or subject variant IL-7sdescribed herein.

In yet another, provided herein is a method of inducing Ki67 expressionin a lymphocyte in a subject comprising administering to the subject acomposition that includes any of the subject dimeric fusion proteins,subject heterodimeric fusion proteins, or subject variant IL-7sdescribed herein.

In some embodiments, the lymphocyte is a CD4⁺CD45RA⁺ lymphocyte, aCD4⁺CD45RA⁻ lymphocyte, a CD8⁺CD45RA⁺ lymphocyte, a CD8⁺CD45RA⁻lymphocyte, a γδ T cell, a CD56⁺ NK cell, or a CD16⁺ NK cell.

In one aspect, provided herein is method of activating and/or inducingproliferation of a lymphocyte population. Such a method comprisescontacting the lymphocyte population with a composition that includesany of the subject dimeric fusion proteins, subject heterodimeric fusionproteins, or subject variant IL-7s described herein.

In another method, provided herein is a method of activating and/orinducing proliferation of a lymphocyte population in a subject. Themethod comprises administering to the subject a composition thatincludes any of the subject dimeric fusion proteins, subjectheterodimeric fusion proteins, or subject variant IL-7s describedherein.

In some embodiments, the lymphocyte population is a CD45⁺ cellpopulation, a CD3⁺ T cell population, a CD4⁺ T cell population, a CD8⁺ Tcell population, or an NK cell population.

In another aspect, provided herein is a method of increase IFNγ or CD25production in a subject. The method comprises administering to thesubject a composition that includes any of the subject dimeric fusionproteins, subject heterodimeric fusion proteins, or subject variantIL-7s described herein.

In yet another aspect, provided herein is a method of reducing a tumorcomprising contacting the tumor with a composition that includes any ofthe subject dimeric fusion proteins, subject heterodimeric fusionproteins, or subject variant IL-7s described herein.

In one aspect, provided herein is a method of reducing a tumor in asubject in need thereof comprising administering to the subject acomposition that includes any of the subject dimeric fusion proteins,subject heterodimeric fusion proteins, or subject variant IL-7sdescribed herein.

In another aspect, provided herein is method of treating a subjecthaving a cancer, comprising administering to the subject a compositionthat includes any of the subject dimeric fusion proteins, subjectheterodimeric fusion proteins, or subject variant IL-7s describedherein.

In some embodiments of the methods provided herein, the subject is ahuman subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequences for human IL-7 and its receptor.

FIG. 2 depicts the sequences for mouse IL-7 and its receptors tofacilitate investigation of IL-7 fusion proteins described herein inpreclinical studies.

FIG. 3 depicts the sequences for cynomolgus IL-7 and its receptors tofacilitate investigation of IL-7 fusion proteins described herein inpreclinical studies.

FIGS. 4A-4E depict useful pairs of Fc heterodimerization variant sets(including skew and pI variants) that can be used in the IL-7heterodimeric Fc fusion proteins described herein. Variants without acorresponding “monomer 2” are pI variants which can be used alone oneither monomer.

FIG. 5 depict a list of isosteric variant antibody constant regions andtheir respective substitutions. pI_(−) indicates lower pI variants,while pI_(+) indicates higher pI variants. These can be optionally andindependently combined with other heterodimerization variants in theIL-7 heterodimeric Fc fusion proteins described herein (and othervariant types as well, as outlined herein.)

FIG. 6 depicts useful ablation variants that ablate FcγR binding(sometimes referred to as “knock outs” or “KO” variants). Generally,ablation variants are found on both monomers, although in some casesthey may be on only one monomer. Such ablation variants can be used inthe IL-7-Fc fusion proteins (dimeric, including homodimeric andheterodimeric fusion proteins) described herein.

FIG. 7 depicts a number of exemplary domain linkers. In someembodiments, these linkers find use linking an IL-7 monomer to theN-terminus of an Fc chain. In other embodiments, these linkers find uselinking an IL-7 monomer to the C-terminus of an Fc chain.

FIG. 8 shows particularly useful embodiments of “non-cytokine”components of the IL-7 fusions of the invention. FIG. 8A finds use inmonovalent IL-7-Fc fusion formats of the IL-7-Fc fusion proteins,including, but not limited to, (IL-7)₁-Fc, (IL-7)₁-L-Fc, Fc-(IL-7)₁, andFc-L-(IL-7)₁. In some embodiments of the IL-7-Fc fusion formats, IL-7 isattached to the N-terminus or C-terminus of “monomer 2.” In otherembodiments of the IL-7-Fc fusion formats, IL-7 is attached to theN-terminus or C-terminus of “monomer 1.” FIG. 8B finds use in bivalentIL-7-Fc fusion formats of the IL-7-Fc fusion proteins, including, butnot limited to, (IL-7)₂-Fc, (IL-7)₂-L-Fc, Fc-(IL-7)₂, and Fc-L-(IL-7)₂.

FIG. 9 shows the sequences of several useful homodimeric IL-7 fusionbackbones based on human IgG, without the cytokine sequences. Thesesequences can be used with any of the IL-7-Fc fusion proteins describedherein that utilize a homodimeric Fc region. IL-7-Fc fusion proteinsthat include such backbones in FIG. 9, include a first and secondmonomer with the same backbone. Such IL-7-Fc fusion proteins furtherinclude an IL-7 attached to the N-terminus or C-terminus of eachbackbone. Homodimeric Fc backbone 1 is based on human IgG1 (356E/358Mallotype), and includes the E233P/L234V/L235A/G236del/S267K ablationvariants and C220S. Homodimeric Fc backbone 2 is based on human IgG1(356D/358L allotype), and includes the E233P/L234V/L235A/G236del/S267Kablation variants and C220S. Homodimeric Fc backbone 3 is based on humanIgG4, and the S228P (according to EU numbering; S241P in Kabat) variantthat ablates Fab arm exchange (as is known in the art). Homodimeric Fcbackbone 4 is based on human IgG2, and includes the S267K ablationvariant. Alternative formats for homodimeric backbone 4 can includeC219S and/or C220S. It should be noted that for C-terminal Fc fusionformats, the backbones may further comprise deletion of K447 on one orboth chains. Furthermore, any of these sequences can include Xtendsubstitutions (M428L/N434S).

FIGS. 10A-10C show the sequences of several useful heterodimeric IL-7fusion backbones based on human IgG, without the cytokine sequences. Theheterodimeric IL-7 fusion backbone sequences can be used with anyIL-7-Fc fusion protein described herein that include a heterodimeric Fcregion (e.g., monovalent IL-7-Fc fusion proteins). Subject monovalentIL-7-Fc fusion proteins that includes such backbone include a firstmonomer that includes a “monomer 1” backbone and a second monomer thatincludes a “monomer 2” backbone. In preferred embodiments, the secondmonomer further includes an IL-7 attached to the “monomer 2” backbone.Heterodimeric Fc backbone 1 is based on human IgG1 (356E/358M allotype),and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants and C220S on bothchains. Heterodimeric Fc backbone 2 is based on human IgG1 (356E/358Mallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants and C220S on bothchains. Heterodimeric Fc backbone 3 is based on human IgG1 (356E/358Mallotype), and includes the L368E/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants and C220S on bothchains. Heterodimeric Fc backbone 4 is based on human IgG1 (356E/358Mallotype), and includes the K360E/Q362E/T411E skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the D401K skew variant on a second heterodimeric Fc chain, and theE233P/L234V/L235A/G236del/S267K ablation variants and C220S on bothchains. Heterodimeric Fc backbone 5 is based on human IgG1 (356D/358Lallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants and C220S on bothchains. Heterodimeric Fc backbone 6 is based on human IgG1 (356E/358Mallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants, N297A variantthat removes glycosylation, and C220S on both chains. Heterodimeric Fcbackbone 7 is based on human IgG1 (356E/358M allotype), and includes theL368D/K370S skew variants and the Q295E/N384D/Q418E/N421D pI variants ona first heterodimeric Fc chain, the S364K/E357Q skew variants on asecond heterodimeric Fc chain, and the E233P/L234V/L235A/G236del/S267Kablation variants, N297S variant that removes glycosylation, and C220Son both chains. Heterodimeric Fc backbone 8 is based on human IgG4, andincludes the L368D/K370S skew variants and the Q295E/N384D/Q418E/N421DpI variants on a first heterodimeric Fc chain, the S364K/E357Q skewvariants on a second heterodimeric Fc chain, and the S228P (according toEU numbering, S241P in Kabat) variant that ablates Fab arm exchange (asis known in the art) on both chains. Heterodimeric Fc backbone 9 isbased on human IgG2, and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain.Heterodimeric Fc backbone 10 is based on human IgG2, and includes theL368D/K370S skew variants and the Q295E/N384D/Q418E/N421D pI variants ona first heterodimeric Fc chain, the S364K/E357Q skew variants on asecond heterodimeric Fc chain, and the S267K ablation variant on bothchains. Alternative formats for heterodimeric Fc backbones 9 and 10 caninclude C220S and/or C219S (in the case of a backbone based on IgG2).Heterodimeric Fc backbone 11 is based on human IgG1 (356E/358Mallotype), and includes the L368D/K370S skew variants and theQ295E/N384D/Q418E/N421D pI variants on a first heterodimeric Fc chain,the S364K/E357Q skew variants on a second heterodimeric Fc chain, andthe E233P/L234V/L235A/G236del/S267K ablation variants, M428L/N434S Xtendvariants, and C220S on both chains. Heterodimeric Fc backbone 12 isbased on human IgG1 (356E/358M allotype), and includes the L368D/K370Sskew variants on a first heterodimeric Fc chain, the S364K/E357Q skewvariants and P217R/P228R/N276K pI variants on a second heterodimeric Fcchain, and the E233P/L234V/L235A/G236del/S267K ablation variants andC220S on both chains. It should be noted that for C-terminal IL-7-Fcfusion formats, the backbones may further comprise deletion of K447 onone or both chains.

In some embodiments, the IL-7-Fc fusion protein described herein includean Fc fusion backbone sequence that is 90, 95, 98 and 99% identical (asdefined herein) to a Fc fusion backbone sequence in FIG. 9 or 10, and/orcontain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acidsubstitutions (as compared to the “parent” of the Figure, which, as willbe appreciated by those in the art, already contain a number of aminoacid modifications as compared to the parental human IgG1 (or IgG2 orIgG4, depending on the backbone). That is, the recited backbones maycontain additional amino acid modifications (generally amino acidsubstitutions) in addition or as an alternative to the skew, pI andablation variants contained within the backbones of FIGS. 9 and 10.

FIGS. 11A-11B depicts illustrative formats for IL-7 fusions in thebivalent N-terminal IL-7-Fc fusion category. One such format in thiscategory is the (IL-7)₂-Fc format (cartoon schematic depicted in FIG.11A) which includes two identical monomers, each monomer comprising anIL-7 covalently attached to the N-terminus of a homodimeric Fc chain.Another such format in this category is the (IL-7)₂-L-Fc format (cartoonschematic depicted in FIG. 11B) which comprises two identical monomers,each monomer comprising an IL-7 monomer covalently attached to theN-terminus of a homodimeric Fc chain via a domain linker.

FIG. 12 depicts the sequences for XENP27088, an illustrative IL-7 fusionof the bivalent N-terminal IL-7 fusion in the (IL-7)₂-Fc format. IL-7sequences are italicized and slashes (/) indicate the border(s) betweenIL-7 monomer and Fc regions. It should be noted that while the IL-7sequences are wild-type, the IL-7 fusions can utilize an IL-7 sequencesthat are 90, 95, 98 and 99% identical (as defined herein), and/orcontain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acidsubstitutions, including substitutions to modulate affinity/potencyand/or reduce heterogeneity (e.g., those in FIGS. 29 and 30).Additionally, any of these sequences can include Xtend substitutions(M428L/N434S).

FIG. 13 depicts the sequences for XENP27089, an illustrative IL-7 fusionof the bivalent N-terminal IL-7 fusion in the (IL-7)₂-L-Fc format. IL-7sequences are italicized, domain linkers are double underlined (althoughas will be appreciated by those in the art, the domain linkers can bereplaced by other domain linkers including, but not limited to, thosedepicted in FIG. 7), and slashes (/) indicate the border(s) between IL-7monomer, linkers, and Fc regions. It should be noted that while the IL-7sequences are wild-type, the IL-7 fusions can utilize variant IL-7sequences that are 90, 95, 98 and 99% identical (as defined herein),and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional aminoacid substitutions, including substitutions to modulate affinity/potencyand/or reduce heterogeneity (e.g., those in FIGS. 29 and 30).Additionally, any of these sequences can include Xtend substitutions(M428L/N434S).

FIGS. 14A-14B depicts illustrative formats for IL-7 fusions in themonovalent N-terminal IL-7-Fc fusion category. One such format of thiscategory is the (IL-7)₁-Fc format (depicted in FIG. 14A), which includesa first monomer that includes an IL-7 monomer covalently attached to theN-terminus of a first heterodimeric Fc chain, and a second monomer thatincludes a complementary second heterodimeric Fc chain that is “Fc-only”or “empty-Fc.” Another such format of this category is the (IL-7)₁-L-Fcformat (depicted in FIG. 14B), which includes a first monomer thatincludes an IL-7 monomer covalently attached to the N-terminus of afirst heterodimeric Fc chain via a domain linker, and a second monomerthat includes a complementary second heterodimeric Fc chain that is“Fc-only” or “empty-Fc.”

FIG. 15 depicts the sequences for XENP27079, an illustrative IL-7 fusionof the monovalent N-terminal IL-7 fusion in the (IL-7)₁-Fc format. IL-7sequences are italicized and slashes (/) indicate the border(s) betweenIL-7 monomer and Fc regions. It should be noted that while the IL-7sequences are wild-type, the IL-7 fusions can utilize IL-7 sequencesthat are 90, 95, 98 and 99% identical (as defined herein), and/orcontain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acidsubstitutions, including substitutions to modulate affinity/potencyand/or reduce heterogeneity (e.g., those in FIGS. 29 and 30).Additionally, any of these sequences can include Xtend substitutions(M428L/N434S).

FIG. 16 depicts the sequences for XENP27080, an illustrative IL-7 fusionof the monovalent N-terminal IL-7 fusion in the (IL-7)₁-L-Fc format.IL-7 sequences are italicized, domain linkers are double underlined(although as will be appreciated by those in the art, the domain linkerscan be replaced by other domain linkers including, but not limited tothose depicted in FIG. 7), and slashes (/) indicate the border(s)between IL-7 monomer, linkers, and Fc regions. It should be noted thatwhile the IL-7 sequences are wild-type, the IL-7 fusions can utilizeIL-7 sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additionalamino acid substitutions, including substitutions to modulateaffinity/potency and/or reduce heterogeneity (e.g., those in FIGS. 29and 30). Additionally, any of these sequences can include Xtendsubstitutions (M428L/N434S).

FIGS. 17A-17B depicts illustrative formats for IL-7 fusions in thebivalent C-terminal IL-7-Fc fusion category. One such format of thiscategory is the Fc-(IL-7)₂ format (depicted in FIG. 17A), which includestwo identical monomers, each monomer includes an IL-7 monomer covalentlyattached to the C-terminus of a homodimeric Fc chain. Another suchformat of this category is the Fc-L-(IL-7)₂ format (depicted in FIG.17B) which includes two identical monomers, where each monomer includesan IL-7 monomer covalently attached to the C-terminus of a homodimericFc chain via a domain linker.

FIG. 18 depicts the sequences for XENP27090, an illustrative IL-7 fusionof the bivalent C-terminal IL-7 fusion in the Fc-L-(IL-7)₂ format. IL-7sequences are italicized, domain linkers are double underlined (althoughas will be appreciated by those in the art, the domain linkers can bereplaced by other domain linkers including, but not limited to thosedepicted in FIG. 7), and slashes (/) indicate the border(s) between IL-7monomer, linkers, and Fc regions. It should be noted that while the IL-7sequences are wild-type, the IL-7 fusions can utilize IL-7 sequencesthat are 90, 95, 98 and 99% identical (as defined herein), and/orcontain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acidsubstitutions, including substitutions to modulate affinity/potencyand/or reduce heterogeneity (e.g., those in FIGS. 29 and 30).Additionally, any of these sequences can include Xtend substitutions(M428L/N434S).

FIGS. 19A-19B depicts illustrative formats for IL-7 fusions in themonovalent C-terminal IL-7-Fc fusion category. One such format of thiscategory is the Fc-(IL-7)₁ format (depicted in FIG. 19A) which includesa first monomer that an IL-7 monomer covalently attached to theC-terminus of a first heterodimeric Fc chain, and a second monomer thatincludes a complementary second heterodimeric Fc chain that is “Fc-only”or “empty-Fc”. Another such format of this category is the Fc-L-(IL-7)₁format (depicted in FIG. 19B) which includes a first monomer thatincludes an IL-7 monomer covalently attached to the C-terminus of afirst heterodimeric Fc chain via a domain linker, and a second monomerthat includes a complementary second heterodimeric Fc chain that is“Fc-only” or “empty-Fc.”

FIG. 20 depicts the sequences for XENP27083, an illustrative IL-7 fusionof the monovalent C-terminal IL-7 fusion in the Fc-L-(IL-7)₁ format.IL-7 sequences are italicized, domain linkers are double underlined(although as will be appreciated by those in the art, the domain linkerscan be replaced by other domain linkers including, but not limited tothose depicted in FIG. 7), and slashes (/) indicate the border(s)between IL-7 monomer, linkers, and Fc regions. It should be noted thatwhile the IL-7 sequences are wild-type, the IL-7 fusions can utilize anIL-7 sequences that are 90, 95, 98 and 99% identical (as definedherein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additionalamino acid substitutions, including substitutions to modulateaffinity/potency and/or reduce heterogeneity (e.g., those in FIGS. 29and 30). Additionally, any of these sequences can include Xtendsubstitutions (M428L/N434S).

FIGS. 21A-21F depict induction of STAT5 phosphorylation by IL-7-Fcfusion proteins in the various formats (as well as recombinant humanIL-7 control) on A) CD4⁺CD45RA⁺, B) CD4⁺CD45RA⁻, C) CD8⁺CD45RA⁺, D)CD8⁺CD45RA⁻, E) CD56⁺ NK cells, and F) Tregs. The data show that each ofthe prototype IL-7-Fc fusions were active in inducing STAT5phosphorylation on various lymphocyte populations and that theparticular format of the IL-7-Fc fusions did not impact on the potencyof STAT5 signaling. Notably, the data show that the IL-7-Fc fusions weremore potent than recombinant IL-7. Additionally, the data show that CD4⁺T cells were the most potent responders to recombinant IL-7 and thevarious IL-7-Fc fusions.

FIGS. 22A-22G depicts induction of Ki67 by IL-7-Fc fusion proteins inthe various formats (as well as recombinant human IL-7 control) on A)CD4⁺CD45RA⁺, B) CD4⁺CD45RA⁻, C) CD8⁺CD45RA⁺, D) CD8⁺CD45RA⁻, E) γδ Tcells, F) CD56⁺ NK cells, and G) CD16⁺ NK cells. The data show that eachof the prototype IL-7-Fc fusions were active in inducing Ki67 on variouslymphocyte populations and that the particular format of the IL-7-Fcfusions did not impact on the potency of proliferative activity.Notably, the data show that the IL-7-Fc fusions were more potent thanrecombinant IL-7. Additionally, the data show that CD4⁺ T cells were themost potent responders to recombinant IL-7 and the various IL-7-Fcfusions.

FIG. 23 depicts the sequences for XENP16432, anti-PD-1 mAb based onnivolumab and IgG1 backbone with E233P/L234V/L235A/G236del/S267Kablation variant.

FIGS. 24A-24H depict the body weight (as a percentage of initial bodyweight) of huPBMC-engrafted NSG mice (dosed with the indicated testarticles) on A) Day 3, B) Day 6, C) Day 10, D) Day 13, E) Day 17, F) Day20, G) Day 27, and H) over time. XENP27080 significantly enhanced bodyweight loss on Days 13 and 17 in comparison to checkpoint blockade byXENP16432 (statistics performed on data using unpaired t-test), andresulted in death of 2 mice by Day 20.

FIGS. 25A-25F depict A) CD45 cell, B) CD3⁺ T cell, C) CD4⁺ T cell, D)CD8⁺ T cell, E) NK cell counts as well as CD4⁺ T cell to CD8⁺ T cellratio in huPBMC-engrafted NSG mice on Day 7 following dosing with theindicated test articles. The IL-7-Fc fusion XENP27080 had significantlyenhanced expansion of CD45⁺, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells,and NK cells by Day 7 in comparison to PBS control (statistics wereperformed on log-transformed data using unpaired t-test).

FIG. 26A-26F depict A) CD45 cell, B) CD3⁺ T cell, C) CD4⁺ T cell, D)CD8⁺ T cell, E) NK cell counts as well as CD4⁺ T cell to CD8⁺ T cellratio in huPBMC-engrafted NSG mice on Day 10 following dosing with theindicated test articles (statistics were performed on log-transformeddata using unpaired t-test).

FIG. 27A-27F depict A) CD45 cell, B) CD3⁺ T cell, C) CD4⁺ T cell, D)CD8⁺ T cell, E) NK cell counts as well as CD4⁺ T cell to CD8⁺ T cellratio in huPBMC-engrafted NSG mice on Day 14 following dosing with theindicated test articles. XENP27080 had significantly enhanced expansionof CD45⁺, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, and NK cells by Day14 in comparison to both PBS control and checkpoint blockade byXENP16432 (statistics performed on log-transformed data using unpairedt-test).

FIGS. 28A-28B depicts serum concentration of A) IFNγ and B) CD25 inhuPBMC-engrafted NSG mice on Days 7, 10, and 14 following dosing withthe indicated test articles. The data show enhanced secretion of thecytokines over the duration of the study.

FIGS. 29A and 29B depict sequences for illustrative IL-7 variantsengineered with the aim to reduce heterogeneity. Modified amino acidsare underlined and in bold. It should be noted that each of thesubstitutions depicted in this Figure can be used alone or incombination with any other substitutions depicted herein, includingsubstitutions to modulate affinity/potency and/or reduce heterogeneity.Although the illustrative sequences as depicted in FIG. 29 includesubstitutions of the asparagine (N) at positions 70, 91, and/or 116 withalanine (A), glutamine (Q), or aspartic acid (D), the asparagine atpositions 70, 91, and/or 116 can be substituted with any amino acid toprevent glycosylation. Additionally or alternatively, the threonine atpositions 72 and 93 and the serine at position 118 can be substitutedwith any amino acid other than threonine and serine to preventglycosylation. Additional engineering approaches as known in the art mayalso be used to prevent glycosylation of the IL-7 moiety.

FIGS. 30A-30C depict sequences for illustrative IL-7 variants engineeredwith the aim to reduce binding affinity for IL-7Rα and/or CD132.Modified amino acids are underlined and in bold. It should be noted thateach of the substitutions depicted in this Figure can be used alone orin combination with any other substitutions depicted herein, includingsubstitutions to modulate affinity/potency and/or reduce heterogeneity.

FIGS. 31A-31E depict the sequences of illustrative IL-7 fusions of thebivalent N-terminal IL-7-Fc fusion category in the (IL-7)₂-L-Fc formatcomprising IL-7 variants engineered with the aim to modulateaffinity/potency and/or reduce heterogeneity. IL-7 sequences areitalicized, domain linkers are double underlined (although as will beappreciated by those in the art, the domain linkers can be replaced byother domain linkers including, but not limited to those depicted inFIG. 7), and slashes (/) indicate the border(s) between IL-7 monomer,linkers, and Fc regions. It should be noted that each of the IL-7-Fcfusions can utilize an IL-7 sequence that is 90, 95, 98 and 99%identical (as defined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 additional amino acid substitutions compared to therespective IL-7 sequence depicted, including substitutions to modulateaffinity/potency and/or reduce heterogeneity. Additionally, any of thesesequences can include Xtend substitutions (M428L/N434S).

FIGS. 32A-32C depict the sequences of illustrative IL-7 fusions of themonovalent N-terminal IL-7-Fc fusion category in the (IL-7)₁-Fc formatcomprising IL-7 variants engineered with the aim to modulateaffinity/potency and/or reduce heterogeneity. IL-7 sequences areitalicized and slashes (/) indicate the border(s) between IL-7 monomerand Fc regions. It should be noted that each of the IL-7-Fc fusions canutilize an IL-7 sequence that is 90, 95, 98 and 99% identical (asdefined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10additional amino acid substitutions compared to the respective IL-7sequence depicted, including substitutions to modulate affinity/potencyand/or reduce heterogeneity. Additionally, any of these sequences caninclude Xtend substitutions (M428L/N434S).

FIGS. 33A-33C depicts the sequences of illustrative IL-7 fusions of themonovalent N-terminal IL-7-Fc fusion category in the (IL-7)₁-L-Fc formatcomprising IL-7 variants engineered with the aim to modulateaffinity/potency and/or reduce heterogeneity. IL-7 sequences areitalicized, domain linkers are double underlined (although as will beappreciated by those in the art, the domain linkers can be replaced byother domain linkers including, but not limited to those depicted inFIG. 7), and slashes (/) indicate the border(s) between IL-7 monomer,linkers, and Fc regions. It should be noted that each of the IL-7fusions can utilize an IL-7 sequence that is 90, 95, 98 and 99%identical (as defined herein), and/or contain from 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 additional amino acid substitutions compared to therespective IL-7 sequence depicted, including substitutions to modulateaffinity/potency and/or reduce heterogeneity. Additionally, any of thesesequences can include Xtend substitutions (M428L/N434S).

FIG. 34 depicts the heterogeneity of illustrative (IL-7)₂-L-Fc fusionsas determined by CEF. Bands for XENP28759, XENP28760, XENP28766, andXENP28770 were less diffuse than band for XENP27089 indicating areduction in heterogeneity.

FIG. 35 depicts the maximum BLI-response of binding by (IL-7)₂-L-Fcfusions comprising variant IL-7 to IL-7Rα as determined by Octet, aswell as response ratio relative to (IL-7)₂-L-Fc fusion comprising WTIL-7 (XENP27089). The data show that the engineered IL-7-Fc fusionsexhibit a range of binding capacity for IL-7Rα with several variantsdemonstrating drastically reduced binding in comparison to WT IL-7-Fcfusion. Notably, several of the IL-7-Fc fusions comprising IL-7 variantsengineered for reduced heterogeneity also demonstrated reduced binding.

FIGS. 36A-36F depict induction of STAT5 phosphorylation (as indicated bypSTAT5 MFI on various lymphocyte populations) by illustrative IL-7fusions of the monovalent N-terminal IL-7-Fc fusion category in the(IL-7)₁-Fc format comprising IL-7 variants engineered with the aim tomodulate affinity/potency on A) CD4⁺ T cells, B) CD8⁺ T cells, C) CD4⁺memory T cells, D) CD4⁺ naive T cells, E) CD8⁺ memory T cells, and F)CD8⁺ naive T cells. The data show that (IL-7)₁-Fc fusions comprisingD74N and K81E both demonstrated reduced potency compared to WT (albeit,a much greater reduction in potency by K81E), and combining the twosubstitutions D74N/K81E proved synergistic and demonstrated the greatestreduction in potency. Furthermore, the data show that the IL-7-Fcfusions were generally more potent on CD4⁺ T cells compared to CD8⁺ Tcells. Additionally, the data shows that the variant (IL-7)₁-Fc fusionsshow similar potency in induction of CD4⁺ memory T cells and CD4⁺ naiveT cells, but are more potent in induction of CD8⁺ naïve T cells than ininduction of CD8⁺ memory T cells.

FIGS. 37A-37F depict induction of STAT5 phosphorylation (as indicated bypercentage of various lymphocyte populations that are pSTAT5⁺) byillustrative IL-7 fusions of the monovalent N-terminal IL-7-Fc fusioncategory in the (IL-7)₁-Fc format comprising IL-7 variants engineeredwith the aim to modulate affinity/potency on A) CD4⁺ T cells, B) CD8⁺ Tcells, C) CD4⁺ memory T cells, D) CD4⁺ naive T cells, E) CD8⁺ memory Tcells, and F) CD8⁺ naive T cells.

FIGS. 38A-38F depict induction of STAT5 phosphorylation (as indicated bypSTAT5 MFI on various lymphocyte populations) by illustrative IL-7fusions of the monovalent N-terminal IL-7-Fc fusion category in the(IL-7)₁-L-Fc format comprising IL-7 variants engineered with the aim tomodulate affinity/potency on A) CD4⁺ T cells, B) CD8⁺ T cells, C) CD4⁺memory T cells, D) CD4⁺ naive T cells, E) CD8⁺ memory T cells, and F)CD8⁺ naive T cells. The data show that (IL-7)₁-Fc fusions comprisingD74N and K81E both demonstrated reduced potency compared to WT (albeit,a much greater reduction in potency by K81E), and combining the twosubstitutions D74N/K81E proved synergistic and demonstrated the greatestreduction in potency. Furthermore, the data show that the IL-7-Fcfusions were generally more potent on CD4⁺ T cells compared to CD8⁺ Tcells. Additionally, the data shows that the variant (IL-7)₁-Fc fusionsshow similar potency in induction of CD4⁺ memory T cells and CD4⁺ naiveT cells, but are more potent in induction of CD8⁺ naïve T cells than ininduction of CD8⁺ memory T cells. Notably, the data shows that thelinker does not impact on the potency of the IL-7-Fc fusions.

FIGS. 39A-39F depict induction of STAT5 phosphorylation (as indicated bypercentage of various lymphocyte populations that are pSTAT5⁺) byillustrative IL-7 fusions of the monovalent N-terminal IL-7-Fc fusioncategory in the (IL-7)₁-L-Fc format comprising IL-7 variants engineeredwith the aim to modulate affinity/potency on A) CD4⁺ T cells, B) CD8⁺ Tcells, C) CD4⁺ memory T cells, D) CD4⁺ naive T cells, E) CD8⁺ memory Tcells, and F) CD8⁺ naive T cells.

DETAILED DESCRIPTION I. Overview

Provided herein are dimeric IL-7-Fc fusion proteins that include Fcdomains and one or more IL-7s. As discussed herein, such IL-7-Fc fusionproteins exhibit IL-7 biological activity and long serum half-lives. Dueto the long serum half-lives, the fusion proteins advantageously do notrequire high doses for use in treatments, thereby minimizing anypotential systemic toxicity associated with increased IL-7 levels. Thedimeric IL-7-Fc fusion proteins can be used for applications whereincreased IL-7 activity is useful, for example, for increasing theproliferation of lymphocyte populations in mounting an anti-tumorresponse in a subject in need thereof.

II. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “IL-7,” “Interleukin-7,” and “IL7” herein is meant a hematopoieticgrowth factor that binds to IL-7 receptor and is capable of stimulatingcell growth and proliferation in the lymphoid lineage (e.g., B cells, Tcells and NK cells). IL-7 receptor includes two subunits IL-7 receptor-αsubunit (; IL-7Rα or CD127) and a common-γ chain receptor (CD132).Sequences of various IL-7s and corresponding IL-7 receptors are shown inFIGS. 1-3. Sequences of exemplary wildtype human precursor and matureIL-7, as well as the IL-7 receptor subunits are included in FIG. 1.

By “ablation” herein is meant a decrease or removal of binding and/oractivity. Thus for example, “ablating FcγR binding” means the Fc regionamino acid variant has less than 50% starting binding as compared to anFc region not containing the specific variant, with more than70-80-90-95-98% loss of binding being preferred, and in general, withthe binding being below the level of detectable binding in a Biacoreassay. Of particular use in the ablation of FcγR binding are those shownin FIG. 6. However, unless otherwise noted, the Fc monomers of theinvention retain binding to the FcRn.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity. As is discussed herein, some embodiments ablate ADCCactivity entirely.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g., the 20 amino acids that have codonsin DNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y or 272Y refers to a variant polypeptide, in this case an Fcvariant, in which the glutamic acid at position 272 is replaced withtyrosine. For clarity, a protein which has been engineered to change thenucleic acid coding sequence but not to change the starting amino acid(for example exchanging CGG (encoding arginine) to CGA (still encodingarginine) to increase host organism expression levels) is not an “aminoacid substitution”; that is, despite the creation of a new gene encodingthe same protein, if the protein has the same amino acid at theparticular position that it started with, it is not an amino acidsubstitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid residue or sequence at a particular positionin a parent polypeptide sequence. For example, -233E designates aninsertion of glutamic acid after position 233 and before position 234.Additionally, -233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid residue or sequence at a particular position ina parent polypeptide sequence. For example, E233-, E233#, E233( ),E233_, or E233del designates a deletion of glutamic acid at position233. Additionally, EDA233- or EDA233# designates a deletion of thesequence GluAspAla that begins at position 233.

By “variant protein”, “protein variant”, or “variant” as used herein ismeant a protein that differs from that of a parent protein by virtue ofat least one modification. Protein variant may refer to the proteinitself, a composition comprising the protein, the amino acid sequencethat encodes it, or the DNA sequence that encodes it. Preferably, theprotein variant has at least one amino acid modification compared to theparent protein, e.g. from about one to about seventy amino acidmodifications, and preferably from about one to about five amino acidmodifications compared to the parent. The modification can be anaddition, deletion, or substitution. As described below, in someembodiments the parent protein, for example an Fc parent polypeptide, isa human wild type sequence, such as the Fc region from IgG1, IgG2, IgG3or IgG4. The protein variant sequence herein will preferably possess atleast about 80% identity with a parent protein sequence, and mostpreferably at least about 90% identity, more preferably at least about95-98-99% identity. “Variant,” as used herein can also refer toparticular amino acid modifications (e.g., substitutions, deletions,insertions) in a variant protein (e.g., a variant Fc domain), forexample, heterodimerization variants, ablation variants, FcKO variants,etc., as disclosed in Section III below.

As used herein, by “protein” is meant at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. When a biologically functional molecule comprises two or moreproteins, each protein may be referred to as a “monomer” or as a“subunit; and the biologically functional molecule may be referred to asa “complex.”

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein with respect toan IgG domain is meant an amino acid modification that is not isotypic.For example, because none of the IgGs comprise a serine at position 434,the substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof)is considered a non-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” or “Fc ligand” as used herein is meant a molecule,preferably a polypeptide, from any organism that binds to the Fc regionof an IgG antibody to form an Fc/Fc ligand complex. Fc ligands includebut are not limited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannanbinding lectin, mannose receptor, staphylococcal protein A,streptococcal protein G, and viral FcγR. Fc ligands also include Fcreceptor homologs (FcRH), which are a family of Fc receptors that arehomologous to the FcγRs (Davis et al., 2002, Immunological Reviews190:123-136, entirely incorporated by reference). Fc ligands may includeundiscovered molecules that bind Fc. Particular IgG Fc ligands are FcRnand Fc gamma receptors.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin (β2-microglobulin) and the heavy chain is encodedby the FcRn gene. Unless otherwise noted herein, FcRn or an FcRn proteinrefers to the complex of FcRn heavy chain with β2-microglobulin. Avariety of Fc variants can be used to increase binding to the FcRn, andin some cases, to increase serum half-life. In general, unless otherwisenoted, the Fc monomers of the invention retain binding to the FcRn (and,as noted below, can include amino acid variants to increase binding tothe FcRn).

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide (i.e., a wildtypepolypeptide), or a variant or engineered version of a naturallyoccurring polypeptide. Parent polypeptide may refer to the polypeptideitself, compositions that comprise the parent polypeptide, or the aminoacid sequence that encodes it.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody, in someinstances, excluding all of the first constant region immunoglobulindomain (e.g., CH1) or a portion thereof, and in some cases, optionallyincluding all or part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3), and optionally all ora portion of the hinge region between CH1 (Cγ1) and CH2 (Cγ2). Thus, insome cases, the Fc domain includes, from N- to C-terminus, CH2-CH3 andhinge-CH2-CH3. In some embodiments, the Fc domain is that from IgG1,IgG2, IgG3 or IgG4, with IgG1 hinge-CH2-CH3 and IgG4 hinge-CH2-CH3finding particular use in many embodiments. Additionally, in certainembodiments, wherein the Fc domain is a human IgG1 Fc domain, the hingeincludes a C220S amino acid substitution. Furthermore, in someembodiments where the Fc domain is a human IgG4 Fc domain, the hingeincludes a S228P amino acid substitution. Although the boundaries of theFc region may vary, the human IgG heavy chain Fc region is usuallydefined to include residues E216, C226, or A231 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. In some embodiments, as is more fully described below, aminoacid modifications are made to the Fc region, for example to alterbinding to one or more FcγR or to the FcRn.

As will be appreciated by those in the art, the exact numbering andplacement of the heavy constant region domains can be different amongdifferent numbering systems. A useful comparison of heavy constantregion numbering according to EU and Kabat is as below, see Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85 and Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda, entirelyincorporated by reference.

TABLE 1 EU Numbering Kabat Numbering CH1 118-215 114-223 Hinge 216-230226-243 CH2 231-340 244-360 CH3 341-447 361-478

“Fc variant” or “variant Fc” as used herein is meant a proteincomprising an amino acid modification in an Fc domain. The modificationcan be an addition, deletion, or substitution. The Fc variants of thepresent invention are defined according to the amino acid modificationsthat compose them. Thus, for example, N434S or 434S is an Fc variantwith the substitution for serine at position 434 relative to the parentFc polypeptide, wherein the numbering is according to the EU index.Likewise, M428L/N434S defines an Fc variant with the substitutions M428Land N434S relative to the parent Fc polypeptide. The identity of the WTamino acid may be unspecified, in which case the aforementioned variantis referred to as 428L/434S. It is noted that the order in whichsubstitutions are provided is arbitrary, that is to say that, forexample, 428L/434S is the same Fc variant as 434S/428L, and so on. Forall positions discussed herein that relate to antibodies or derivativesand fragments thereof (e.g., Fc domains), unless otherwise noted, aminoacid position numbering is according to the EU index. The “EU index” or“EU index as in Kabat” or “EU numbering” scheme refers to the numberingof the EU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA63:78-85, hereby entirely incorporated by reference). The modificationcan be an addition, deletion, or substitution.

By “fusion protein” as used herein is meant covalent joining of at leasttwo proteins or protein domains. Fusion proteins may comprise artificialsequences, e.g. a domain linker, an Fc domain (e.g., a variant Fcdomain), an IL-7 (e.g., a variant IL-7), etc. as described herein. By“Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a domainlinker, as described herein) to one or more different protein domains.Accordingly, an “IL-7-Fc fusion” includes an Fc domain linked(optionally through a domain linker) to an IL-7, as described herein. Insome instances, two Fc fusion proteins can form a homodimeric Fc fusionprotein or a heterodimeric Fc fusion protein. In some embodiments, onemonomer of the heterodimeric IL-7-Fc fusion protein includes an Fcdomain alone (e.g., an “empty Fc domain”) and the other monomer is an Fcfusion, comprising an IL-7, as outlined herein. In other embodiments,both the first and second monomers are Fc fusion proteins that includean Fc domain and an IL-7.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for numbering of antibodydomains (e.g., a CH1, CH2, CH3 or hinge domain).

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve, create, and/or enhancethe ability to “match” to form heterodimers. For example, if some pIvariants are engineered into monomer A (e.g. making the pI higher), thensteric variants that are “charge pairs” that can be utilized as well donot interfere with the pI variants, e.g. the charge variants that make apI higher are put on the same “strand” or “monomer” to preserve bothfunctionalities. Similarly, for “skew” variants that come in pairs of aset as more fully outlined below, the skilled artisan will consider pIin deciding into which strand or monomer that incorporates one set ofthe pair will go, such that pI separation is maximized using the pI ofthe skews as well.

By “wild type,” “wildtype” or WT” herein is meant an amino acid sequenceor a nucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The IL-7-Fc fusion proteins and variant IL-7s provided herein aregenerally isolated or recombinant. “Isolated,” when used to describe thevarious polypeptides disclosed herein, means a polypeptide that has beenidentified and separated and/or recovered from a cell or cell culturefrom which it was expressed. Ordinarily, an isolated polypeptide will beprepared by at least one purification step. An “isolated protein,”refers to a protein which is substantially free of other proteins from acell culture such as host cell proteins. “Recombinant” means theproteins are generated using recombinant nucleic acid techniques inexogeneous host cells.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Publ. App. No. 20160244525, hereby incorporatedby reference.

The degree of identity between an amino acid sequence provided herein(“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

In some embodiments, two or more amino acid sequences are at least 50%,60%, 70%, 80%, or 90% identical. In some embodiments, two or more aminoacid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

By “fused” or “covalently linked” is herein meant that the components(e.g., an IL-7 and an Fc domain) are linked by peptide bonds, eitherdirectly or indirectly via domain linkers, outlined herein.

The strength, or affinity, of specific binding can be expressed in termsof dissociation constant (KD) of the interaction, wherein a smaller KDrepresents greater affinity and a larger KD represents lower affinity.Binding properties can be determined by methods well known in the artsuch as bio-layer interferometry and surface plasmon resonance basedmethods. One such method entails measuring the rates of antigen-bindingsite/antigen or receptor/ligand complex association and dissociation,wherein rates depend on the concentration of the complex partners, theaffinity of the interaction, and geometric parameters that equallyinfluence the rate in both directions. Thus, both the association rate(ka) and the dissociation rate (kd) can be determined, and the ratio ofkd/ka is equal to the dissociation constant KD (See Nature 361:186-187(1993) and Davies et al. (1990) Annual Rev Biochem 59:439-473).

Specific binding for a particular molecule or an epitope can beexhibited, for example, by a molecule (e.g., IL-7) having a KD for itsbinding partner (e.g., IL-7 receptor) of at least about 10-4 M, at leastabout 10-5 M, at least about 10-6 M, at least about 10-7 M, at leastabout 10-8 M, at least about 10-9 M, alternatively at least about 10-10M, at least about 10-11 M, at least about 10-12 M, or greater.Typically, an antigen binding molecule that specifically binds anantigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-,10,000- or more times greater for a control molecule relative to theantigen or epitope.

III. Dimeric Fc Fusion Proteins

In some aspects, provided herein are dimeric IL-7-Fc fusion proteinsthat include a first monomer that includes a first Fc domain and a firstIL-7 and a second monomer that includes a second Fc domain andoptionally a second IL-7. The IL-7-Fc fusion proteins are based on theself-assembling nature of the two Fc domains on each monomer leading toa dimeric IL-7-Fc fusion proteins. Heterodimeric IL-7-Fc fusion are madeby altering the amino acid sequence of each monomer as more fullydiscussed below.

In one aspect, the dimeric IL-7-Fc fusion protein is a homodimericIL-7-Fc fusion protein. Such homodimeric IL-7-Fc fusion proteins includea first monomer and a second each having an Fc domain with the sameamino acid sequence. In another aspect, the dimeric IL-7-Fc fusionprotein is a heterodimeric Fc fusion protein. Such heterodimeric IL-7-Fcfusion protein include a first monomer and a second monomer, each havingan Fc domain with different amino acid sequences (e.g., a monovalentIL-7-Fc fusion protein). As will be appreciated, discussion herein ofcomponents of the IL-7-Fc fusion proteins encompassed by the presentdisclosure is applicable to both homodimeric and heterodimeric Fc fusionproteins as appropriate, unless otherwise specified.

In some embodiments, the dimeric IL-7-Fc fusion protein includes a firstmonomer and a second monomer. In some embodiments, the dimeric IL-7-Fcfusion protein is a monovalent IL-7 fusion (i.e., includes only oneIL-7). In such embodiments, the first monomer includes an Fc domain andan IL-7 and the second monomer includes an Fc domain alone (i.e., noIL-7, an “empty Fc domain,”). In other embodiments, the dimeric IL-7-Fcfusion is a bivalent Fc fusion (i.e., includes two IL-7s). In suchembodiments, the first and second monomers each include an Fc domain andan IL-7.

The Fc domains can be derived from IgG Fc domains, e.g., IgG1, IgG2,IgG3 or IgG4 Fc domains, with IgG1 Fc domains finding particular use inthe invention. As described herein, IgG1 Fc domains may be used, often,but not always in conjunction with ablation variants to ablate effectorfunction. Similarly, when low effector function is desired, IgG4 Fcdomains may be used.

For any of the dimeric IL-7-Fc fusion proteins described herein, thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDRs and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference). Throughout the present specification, theKabat numbering system is generally used when referring to a residue inthe variable domain (approximately, residues 1-107 of the light chainvariable region and residues 1-113 of the heavy chain variable region)and the EU numbering system for Fc regions (e.g., Kabat et al., supra(1991)).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present IL-7 Fc fusion proteins are theheavy chain domains, including, the constant heavy (CH) domains and thehinge domains. In the context of IgG antibodies, the IgG isotypes eachhave three CH regions. Accordingly, “CH” domains in the context of IgGare as follows: “CH1” refers to positions 118-215 according to the EUindex as in Kabat. “Hinge” refers to positions 216-230 according to theEU index as in Kabat. “CH2” refers to positions 231-340 according to theEU index as in Kabat, and “CH3” refers to positions 341-447 according tothe EU index as in Kabat. As shown in Table 1, the exact numbering andplacement of the heavy chain domains can be different among differentnumbering systems. As shown herein and described below, the pI variantscan be in one or more of the CH regions, as well as the hinge region,discussed below.

By “hinge” or “hinge region” or “antibody hinge region” or“immunoglobulin hinge region” herein is meant the flexible polypeptidecomprising the amino acids between the first and second heavy chainconstant domains of an antibody. Structurally, the IgG CH1 domain endsat EU position 215, and the IgG CH2 domain begins at residue EU position231. Thus for IgG the antibody hinge is herein defined to includepositions 216 (E216 in IgG1) to 230 (P230 in IgG1), wherein thenumbering is according to the EU index as in Kabat. In some embodiments,for example in the context of an Fc region, the hinge (full length or afragment of the hinge) is included, generally referring to positions216-230. As noted herein, pI variants can be made in the hinge region aswell.

In exemplary embodiments of the dimeric IL-7 fusion proteins describedherein, each of the first and second monomers include an Fc domain thathas the formula hinge-CH2-CH3.

In some embodiments described herein, the IL-7-Fc fusion includes afirst monomer that includes an Fc domain and a first IL-7. In certainembodiments, the first IL-7 is directly connected to the Fc domain. Insome embodiments, the C-terminus of the first IL-7 is directly connectedto the N-terminus of the Fc domain. In other embodiments, the N-terminusof the first IL-7 is directly connected to the N-terminus of the Fcdomain. In some embodiments, the N-terminus of the first IL-7 isdirectly connected to the C-terminus of the first Fc domain. In yetother embodiments, the C-terminus of the first IL-7 is directlyconnected to the C-terminus of the Fc domain.

In some embodiments described herein, the dimeric IL-7-Fc fusion alsoincludes a second monomer that includes a second Fc domain and a secondIL-7. In certain embodiments, the second IL-7 is directly connected tothe second Fc domain. In some embodiments, the C-terminus of the secondIL-7 is directly connected to the N-terminus of the second Fc domain. Inother embodiments, the N-terminus of the second IL-7 is directlyconnected to the N-terminus of the Fc domain. In some embodiments, theN-terminus of the second IL-7 is directly connected to the C-terminus ofthe second Fc domain. In yet other embodiments, the C-terminus of thesecond IL-7 is directly connected to the second C-terminus of the Fcdomain.

In some embodiments described herein, the dimeric IL-7-Fc fusionincludes a first monomer that includes an “empty Fc domain” (i.e., an Fcdomain without an IL-7) and a second monomer that includes a second Fcdomain and an IL-7. In certain embodiments, the IL-7 is directlyconnected to the second Fc domain. In some embodiments, the C-terminusof the IL-7 is directly connected to the N-terminus of the second Fcdomain. In other embodiments, the N-terminus of the IL-7 is directlyconnected to the N-terminus of the second Fc domain. In someembodiments, the N-terminus of the IL-7 is directly connected to theC-terminus of the second Fc domain. In yet other embodiments, theC-terminus of the IL-7 is directly connected to the second C-terminus ofthe Fc domain.

In certain embodiments, the IL-7 is connected to the Fc domain by alinker. In certain embodiments, the linker is a domain linker. Usefuldomain linker include, but are not limited to, those disclosed in FIG.7. While any suitable linker can be used, many embodiments utilize aglycine-serine polymer, including for example (GS)n, (GSGGS)n (SEQ IDNO: 1), (GGGGS)n (SEQ ID NO: 2), and (GGGS)n (SEQ ID NO: 3), where n isan integer of at least one (and generally from 0 to 1 to 2 to 3 to 4 to5) as well as any peptide sequence that allows for recombinantattachment of the two domains with sufficient length and flexibility toallow each domain to retain its biological function. In some cases, andwith attention being paid to “strandedness”, as outlined below, thelinker is a charged domain linker.

In certain embodiments, the IL-7 fusion protein includes a firstmonomer, wherein an IL-7 is connected to the Fc domain by a domainlinker. In some embodiments, the C-terminus of the IL-7 is connected tothe N-terminus of the Fc domain by a domain linker. In otherembodiments, the N-terminus of the IL-7 is connected to the N-terminusof the Fc domain by a domain linker. In some embodiments, the N-terminusof the IL-7 is connected to the C-terminus of the IL-7 by a domainlinker. In yet other embodiments, the C-terminus of the IL-7 isconnected to the C-terminus of the Fc domain by a domain linker.

In some embodiments described herein, the dimeric IL-7-Fc fusion alsoincludes a second monomer that includes a second Fc domain and a secondIL-7. In certain embodiments, the second IL-7 is connected to the secondFc domain by a domain linker. In some embodiments, the C-terminus of thesecond IL-7 is connected to the N-terminus of the second Fc domain by adomain linker. In other embodiments, the N-terminus of the second IL-7is connected to the N-terminus of the Fc domain by a domain linker. Insome embodiments, the N-terminus of the second IL-7 is connected to theC-terminus of the second Fc domain by a domain linker. In yet otherembodiments, the C-terminus of the second IL-7 is connected to thesecond C-terminus of the Fc domain by a domain linker.

A. Heterodimerization Variants

In some embodiments, the dimeric IL-7-Fc fusion protein is aheterodimeric IL-7-Fc fusion protein. Such heterodimeric proteinsinclude two different Fc domains (one on each of the first and secondmonomers) that include modifications that facilitate theheterodimerization of the first and second monomers and/or allow forease of purification of heterodimers over homodimers, collectivelyreferred to herein as “heterodimerization variants.” As discussed below,heterodimerization variants can include skew variants (e.g., the “knobsand holes” and “charge pairs” variants described below) as well as “pIvariants” that facilitates the separation of homodimers away fromheterodimers. As is generally described in U.S. Pat. No. 9,605,084,hereby incorporated by reference in its entirety and specifically asbelow for the discussion of heterodimerization variants, usefulmechanisms for heterodimerization include “knobs and holes” (“KIH”) asdescribed in U.S. Pat. No. 9,605,084, “electrostatic steering” or“charge pairs” as described in U.S. Pat. No. 9,605,084, pI variants asdescribed in U.S. Pat. No. 9,605,084, and general additional Fc variantsas outlined in U.S. Pat. No. 9,605,084 and below.

1. Skew Variant

In some embodiments, the heterodimeric IL-7-Fc fusion protein includesskew variants, which are one or more amino acid modifications in a firstFc domain (A) and/or a second Fc domain (B) that favor the formation ofFc heterodimers (Fc dimers that include the first and the second Fcdomain; A-B) over Fc homodimers (Fc dimers that include two of the firstFc domain or two of the second Fc domain; A-A or B-B). Suitable skewvariants are included in the FIG. 29 of US Publ. App. No. 2016/0355608,hereby incorporated by reference in its entirety and specifically forits disclosure of skew variants, as well as in FIG. 4.

One mechanism for skew variants is generally referred to in the art as“knobs and holes,” referring to amino acid engineering that createssteric influences to favor heterodimeric formation and disfavorhomodimeric formation, as described in U.S. Ser. No. 61/596,846, Ridgwayet al., Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol.Biol. 1997 270:26; U.S. Pat. No. 8,216,805, all of which are herebyincorporated by reference in their entirety and specifically for thedisclosure of “knobs and holes” mutations. This is sometime referred toherein as “steric variants.” The figures identify a number of “monomerA-monomer B” pairs that rely on “knobs and holes”. In addition, asdescribed in Merchant et al., Nature Biotech. 16:677 (1998), these“knobs and holes” mutations can be combined with disulfide bonds tofurther favor formation of Fc heterodimers.

An additional mechanism for skew variants that finds use in thegeneration of heterodimers is sometimes referred to as “electrostaticsteering” as described in Gunasekaran et al., J. Biol. Chem.285(25):19637 (2010), hereby incorporated by reference in its entirety.This is sometimes referred to herein as “charge pairs.” In thisembodiment, electrostatics are used to skew the formation towardsheterodimerization. As those in the art will appreciate, these may alsohave an effect on pI, and thus on purification, and thus could in somecases also be considered pI variants. However, as these were generatedto force heterodimerization and were not used as purification tools,they are classified as “skew variants”. These include, but are notlimited to, D221E/P228E/L368E paired with D221R/P228R/K409R (e.g., theseare “monomer” corresponding sets) and C220E/P228E/368E paired withC220R/E224R/P228R/K409R.

In some embodiments, the skew variants advantageously and simultaneouslyfavor heterodimerization based on both the “knobs and holes” mechanismas well as the “electrostatic steering” mechanism. In some embodiments,the heterodimeric IL-7-Fc fusion proteins includes one or more sets ofsuch heterodimerization skew variants. Exemplary skew variants that fallinto this category include: S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L;K370S:S364K/E357Q; or a T366S/L368A/Y407V:T366W (optionally including abridging disulfide, T366S/L368A/Y407V/Y349C:T366W/S354C). These variantscome in “pairs” of “sets.” That is, one set of the pair is incorporatedinto the first monomer and the other set of the pair is incorporatedinto the second monomer. In terms of nomenclature, the pair“S364K/E357Q:L368D/K370S” means that one of the monomers includes an Fcdomain that includes the amino acid substitutions S364K and E357Q andthe other monomer includes an Fc domain that includes the amino acidsubstitutions L368D and K370S; as above, the “strandedness” of thesepairs depends on the starting pI. It should be noted that these sets donot necessarily behave as “knobs in holes” variants, with a one-to-onecorrespondence between a residue on one monomer and a residue on theother. That is, these pairs of sets may instead form an interfacebetween the two monomers that encourages heterodimer formation anddiscourages homodimer formation, allowing the percentage of heterodimersthat spontaneously form under biological conditions to be over 90%,rather than the expected 50% (25 homodimer A/A:50% heterodimer A/B:25%homodimer B/B). Exemplary heterodimerization “skew” variants aredepicted in FIG. 4.

In exemplary embodiments, the heterodimeric IL-7-Fc fusion proteinincludes a S364K/E357Q:L368D/K370S; L368D/K370S:S364K;L368E/K370S:S364K; T411T/E360E/Q362E:D401K; L368D/K370S:S364K/E357L;K370S:S364K/E357Q; or a T366S/L368A/Y407V:T366W (optionally including abridging disulfide, T366S/L368A/Y407V/Y349C:T366W/S354C) “skew” variantamino acid substitution set. In an exemplary embodiment, theheterodimeric IL-7-Fc fusion protein includes a“S364K/E357Q:L368D/K370S” amino acid substitution set.

In some embodiments, the skew variants provided herein can be optionallyand independently incorporated with any other modifications, including,but not limited to, other skew variants (see, e.g., in FIG. 37 of USPubl. App. No. 2012/0149876, herein incorporated by reference,particularly for its disclosure of skew variants), pI variants,isotpypic variants, FcRn variants, ablation variants, etc. into one orboth of the first and second Fc domains of the IL-7-Fc fusion protein.Further, individual modifications can also independently and optionallybe included or excluded from the subject IL-7-Fc fusion proteins.

2. pI (Isoelectric Point) Variants for Heterodimers

In some embodiments, the heterodimeric IL-7-Fc fusion protein includespurification variants that advantageously allow for the separation ofheterodimeric IL-7-Fc fusion proteins from homodimeric proteins.

There are several basic mechanisms that can lead to ease of purifyingheterodimeric proteins. One such mechanism relies on the use of pIvariants which include one or more modifications that affect theisoelectric point of one or both of the monomers of the fusion protein,such that each monomer, and subsequently each dimeric species, has adifferent pI, thus allowing the isoelectric purification of A-A, A-B andB-B dimeric proteins. Alternatively, some formats also allow separationon the basis of size. As is further outlined below, it is also possibleto “skew” the formation of heterodimers over homodimers using skewvariants. Thus, a combination of heterodimerization skew variants and pIvariants find particular use in the subject IL-7 fusion proteinsprovided herein.

Additionally, as more fully outlined below, depending on the format ofthe heterodimeric Fc fusion protein, pI variants can be either containedwithin the constant region and/or Fc domains of a monomer, and/or domainlinkers can be used. In some embodiments, the heterodimeric IL-7-Fcfusion protein includes additional modifications for alternativefunctionalities can also create pI changes, such as Fc, FcRn and KOvariants.

In the embodiments that utilizes pI as a separation mechanism to allowthe purification of heterodimeric IL-7-Fc fusion proteins, amino acidmodifications can be introduced into one or both of the monomers of theheterodimeric IL-7-Fc fusion protein. That is, the pI of one of themonomers (referred to herein for simplicity as “monomer A”) can beengineered away from monomer B, or both monomer A and B can be changed,with the pI of monomer A increasing and the pI of monomer B decreasing.As discussed, the pI changes of either or both monomers can be done byremoving or adding a charged residue (e.g., a neutral amino acid isreplaced by a positively or negatively charged amino acid residue, e.g.,glutamine to glutamic acid), changing a charged residue from positive ornegative to the opposite charge (e.g. aspartic acid to lysine) orchanging a charged residue to a neutral residue (e.g., loss of a charge;lysine to serine). A number of these variants are shown in the figures,including, FIGS. 4 and 5.

Creating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers can be done by usinga “wild type” heavy chain constant region and a variant region that hasbeen engineered to either increase or decrease its pI (wt A:B+ or wtA:B−), or by increasing one region and decreasing the other region(A+:B− or A−:B+).

Thus, in general, a component of some embodiments of the present subjectfusion proteins are amino acid variants in the Fc domains or constantdomain regions that are directed to altering the isoelectric point (pI)of at least one, if not both, of the monomers of a dimeric protein byincorporating amino acid substitutions (“pI variants” or “pIsubstitutions”) into one or both of the monomers. The separation of theheterodimers from the two homodimers can be accomplished if the pIs ofthe two monomers differ by as little as 0.1 pH unit, with 0.2, 0.3, 0.4and 0.5 or greater all finding use in the present invention.

As will be appreciated by those in the art, the number of pI variants tobe included on each or both monomer(s) of a heterodimeric IL-7-Fc fusionprotein to achieve good separation will depend in part on the startingpI of the components. That is, to determine which monomer to engineer orin which “direction” (e.g., more positive or more negative), thesequences of the Fc domains and any IL-7 or linker included in eachmonomer are calculated and a decision is made from there based on thepIs of the monomers. As is known in the art, different Fc domains,linkers and IL-7s will have different starting pIs. In general, asoutlined herein, the pIs are engineered to result in a total pIdifference of each monomer of at least about 0.1 logs, with 0.2 to 0.5being preferred as outlined herein.

In general, as will be appreciated by those in the art, there are twogeneral categories of amino acid modifications that affect pI: thosethat increase the pI of the protein (basic changes) and those thatdecrease the pI of the protein (acidic changes). As described herein,all combinations of these variants can be used: one monomer may includea wild type Fc domain, or a variant Fc domain that does not display asignificantly different pI from wild-type, and the other monomerincludes a Fc domain that is either more basic or more acidic.Alternatively, each monomer may be changed, one to more basic and one tomore acidic.

In the case where pI variants are used to achieve heterodimerization, amore modular approach to designing and purifying heterodimeric IL-7-Fcfusion proteins is provided. Thus, in some embodiments,heterodimerization variants (including skew and pI variants) must beengineered. In addition, in some embodiments, the possibility ofimmunogenicity resulting from the pI variants is significantly reducedby importing pI variants from different IgG isotypes such that pI ischanged without introducing significant immunogenicity (see isotypicvariants below). Thus, an additional problem to be solved is theelucidation of low pI constant domains with high human sequence content,e.g. the minimization or avoidance of non-human residues at anyparticular position. Alternatively or in addition to isotypicsubstitutions, the possibility of immunogenicity resulting from the pIvariants is significantly reduced by utilizing isosteric substitutions(e.g. Asn to Asp; and Gln to Glu).

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in US Publ. App. No. US 2012/0028304 (incorporated byreference in its entirety and specifically for the disclosure of pIvariants that provide additional function), lowering the pI of antibodyconstant domains (including those found in Fc fusions) can lead tolonger serum retention in vivo. These pI variants for increased serumhalf-life also facilitate pI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of Fc fusion proteins, as the ability toeither eliminate, minimize and distinguish when homodimers are presentis significant. Similarly, the ability to reliably test thereproducibility of the heterodimeric Fc fusion protein production isimportant.

Exemplary combinations of pI variants are shown in FIGS. 4 and 5, andFIG. 30 of US Publ. App. No. 2016/0355608, all of which are hereinincorporated by reference in its entirety and specifically for thedisclosure of pI variants. As outlined herein and shown in the figures,these changes are shown relative to IgG1, but all isotypes can bealtered this way, as well as isotype hybrids. In the case where theheavy chain constant domain is from IgG2-4, R133E and R133Q can also beused.

In one embodiment, the heterodimeric IL-7-Fc fusion protein includes amonomer with a variant Fc domain having pI variant modifications295E/384D/418E/421D (Q295E/N384D/Q418E/N421D when relative to humanIgG1). In one embodiment, the heterodimeric IL-7-Fc fusion proteinincludes a monomer with a variant Fc domain having pI variantmodifications 217R/228R/276K (P217R/P228R/N276K when relative to humanIgG1). Additional exemplary pI variant modification that can beincorporated into the Fc domain of a subject are depicted in FIG. 5.

In some embodiments, modifications are made in the hinge of the Fcdomain, including positions 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, and 230 based on EU numbering. Thus, pImutations and particularly substitutions can be made in one or more ofpositions 216-230, with 1, 2, 3, 4 or 5 mutations finding use. Again,all possible combinations are contemplated, alone or with other pIvariants in other domains.

Specific substitutions that find use in lowering the pI of hinge domainsinclude, but are not limited to, a deletion at position 221, anon-native valine or threonine at position 222, a deletion at position223, a non-native glutamic acid at position 224, a deletion at position225, a deletion at position 235 and a deletion or a non-native alanineat position 236. In some cases, only pI substitutions are done in thehinge domain, and in others, these substitution(s) are added to other pIvariants in other domains in any combination.

In some embodiments, mutations can be made in the CH2 region, includingpositions 233, 234, 235, 236, 274, 296, 300, 309, 320, 322, 326, 327,334 and 339, based on EU numbering. It should be noted that changes in233-236 can be made to increase effector function (along with 327A) inthe IgG2 backbone. Again, all possible combinations of these 14positions can be made; e.g., an IL-7-Fc fusion protein may include avariant Fc domain with 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 CH2 pIsubstitutions.

Specific substitutions that find use in lowering the pI of CH2 domainsinclude, but are not limited to, a non-native glutamine or glutamic acidat position 274, a non-native phenylalanine at position 296, anon-native phenylalanine at position 300, a non-native valine atposition 309, a non-native glutamic acid at position 320, a non-nativeglutamic acid at position 322, a non-native glutamic acid at position326, a non-native glycine at position 327, a non-native glutamic acid atposition 334, a non-native threonine at position 339, and all possiblecombinations within CH2 and with other domains.

In this embodiment, the modifications can be independently andoptionally selected from position 355, 359, 362, 384, 389, 392, 397,418, 419, 444 and 447 (EU numbering) of the CH3 region. Specificsubstitutions that find use in lowering the pI of CH3 domains include,but are not limited to, a non-native glutamine or glutamic acid atposition 355, a non-native serine at position 384, a non-nativeasparagine or glutamic acid at position 392, a non-native methionine atposition 397, a non-native glutamic acid at position 419, a non-nativeglutamic acid at position 359, a non-native glutamic acid at position362, a non-native glutamic acid at position 389, a non-native glutamicacid at position 418, a non-native glutamic acid at position 444, and adeletion or non-native aspartic acid at position 447.

3. Isotypic Variants

In addition, some embodiments of the IL-7-Fc fusion proteins providedherein rely on the “importation” of pI amino acids at particularpositions from one IgG isotype into another, thus reducing oreliminating the possibility of unwanted immunogenicity being introducedinto the variants. A number of these are shown in FIG. 21 of US Publ.App. No. 2014/0370013, hereby incorporated by reference, particularlyfor its disclosure of isotypic variants. That is, IgG1 is a commonisotype for therapeutic antibodies for a variety of reasons, includinghigh effector function. However, the heavy constant region of IgG1 has ahigher pI than that of IgG2 (8.10 versus 7.31). By introducing IgG2residues at particular positions into the IgG1 backbone, the pI of theresulting monomer is lowered (or increased) and additionally exhibitslonger serum half-life. For example, IgG1 has a glycine (pI 5.97) atposition 137, and IgG2 has a glutamic acid (pI 3.22); importing theglutamic acid will affect the pI of the resulting protein. As isdescribed below, a number of amino acid substitutions are generallyrequired to significantly affect the pI of the variant Fc fusionprotein. However, it should be noted as discussed below that evenchanges in IgG2 molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid modifications are made,either to reduce the overall charge state of the resulting protein(e.g., by changing a higher pI amino acid to a lower pI amino acid), orto allow accommodations in structure for stability, etc. as is furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant modifications in each monomer of the heterodimercan be seen. As discussed herein, having the pIs of the two monomersdiffer by at least 0.5 can allow separation by ion exchangechromatography or isoelectric focusing, or other methods sensitive toisoelectric point.

4. Calculating pI

The pI of each monomer of the IL-7-Fc fusion protein can depend on thepI of the variant Fc domain and the pI of the total monomer, includingthe variant Fc domain and any IL-7 and/or domain linker included in themonomer. Thus, in some embodiments, the change in pI is calculated onthe basis of the variant Fc domain, using the chart in the FIG. 19 of USPubl. App. No. 2014/0370013, hereby incorporated by reference,particularly for its disclosure of methods of calculating pI. Asdiscussed herein, which monomer to engineer is generally decided by theinherent pI of each monomer.

5. pI Variants that Also Confer Better FcRn In Vivo Binding

In the case where the pI variant(s) decreases the pI of the monomer,such modifications can have the added benefit of improving serumretention in vivo.

Fc regions are believed to have longer half-lives in vivo, becausebinding to FcRn at pH 6 in an endosome sequesters the Fc (Ghetie andWard, 1997 Immunol Today. 18(12): 592-598, entirely incorporated byreference). The endosomal compartment then recycles the Fc to the cellsurface. Once the compartment opens to the extracellular space, thehigher pH, ˜7.4, induces the release of Fc back into the blood. In mice,Dall' Acqua et al. showed that Fc mutants with increased FcRn binding atpH 6 and pH 7.4 actually had reduced serum concentrations and the samehalf-life as wild-type Fc (Dall' Acqua et al. 2002, J. Immunol.169:5171-5180, entirely incorporated by reference). The increasedaffinity of Fc for FcRn at pH 7.4 is thought to forbid the release ofthe Fc back into the blood. Therefore, the Fc modifications that willincrease Fc's half-life in vivo will ideally increase FcRn binding atthe lower pH while still allowing release of Fc at higher pH. The aminoacid histidine changes its charge state in the pH range of 6.0 to 7.4.Thus, it is not surprising to find His residues at important positionsin the Fc/FcRn complex.

B. Other Fc Variants for Additional Functionality

In addition to heterodimerization variants, the subject dimeric IL-7-Fcfusion proteins provided herein (both homodimeric and heterodimeric) mayindependently include Fc modifications that affect functionalityincluding, but not limited to, altering binding to one or more Fcreceptors (e.g., FcγR and FcRn).

FcγR Variants

In one embodiment, the IL-7-Fc fusion proteins includes one or moreamino acid modifications that affect binding to one or more Fcγreceptors (i.e., “FcγR variants”). FcγR variants (e.g., amino acidsubstitutions) that result in increased binding as well as decreasedbinding can be useful. For example, it is known that increased bindingto FcγRIIIa results in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell). Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. FcγR variants that find use in the IL-7 fusionproteins include those listed in U.S. Pat. No. 8,188,321 (particularlyFIG. 41) and U.S. Pat. No. 8,084,582, and US Publ. App. Nos. 20060235208and 20070148170, all of which are expressly incorporated herein byreference in their entirety and specifically for the variants disclosedtherein that affect Fcγ receptor binding. Particular variants that finduse include, but are not limited to, 236A, 239D, 239E, 332E, 332D,239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D,332E/330L, 243A, 243L, 264A, 264V and 299T.

In addition, amino acid substitutions that increase affinity for FcγRIIccan also be independently included in the Fc domain variants outlinedherein. Useful substitutions that for FcγRIIc are described in, forexample, U.S. Pat. Nos. 8,188,321 and 10,113,001, all of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein that affect Fcγ receptorbinding.

FcRn Variants

Further, IL-7-Fc fusion proteins described herein can independentlyinclude Fc substitutions that confer increased binding to the FcRn andincreased serum half-life. Such modifications are disclosed, forexample, in U.S. Pat. No. 8,367,805, hereby incorporated by reference inits entirety, and specifically for Fc substitutions that increasebinding to FcRn and increase half-life. Such modifications include, butare not limited to 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.

Ablation Variants

In some embodiments, the IL-7-Fc fusion protein includes one or moremodifications that reduce or remove the normal binding of the Fc domainto one or more or all of the Fcγ receptors (e.g., FcγR1, FcγRIIa,FcγRIIb, FcγRIIIa, etc.) to avoid additional mechanisms of action. Suchmodifications are referred to as “FcγR ablation variants” or “Fc knockout (FcKO or KO)” variants. In some embodiments, particularly in the useof immunomodulatory proteins, it is desirable to ablate FcγRIIIa bindingto eliminate or significantly reduce ADCC activity such that one of theFc domains comprises one or more Fcγ receptor ablation variants. Theseablation variants are depicted in FIG. 31 of U.S. Pat. No. 10,259,887,which is herein incorporated by reference in its entirety, and each canbe independently and optionally included or excluded, with preferredaspects utilizing ablation variants selected from the group consistingof G236R/L328R, E233P/L234V/L235A/G236del/S239K,E233P/L234V/L235A/G236del/S267K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G and E233P/L234V/L235A/G236del,according to the EU index. In addition, ablation variants of use in thesubject IL-7-Fc fusion proteins are also depicted in FIG. 6. It shouldbe noted that the ablation variants referenced herein ablate FcγRbinding but generally not FcRn binding.

C. Combination of Heterodimeric and Fc Variants

As will be appreciated by those in the art, the Fc modificationsdescribed herein can independently be combined. For example, all of therecited heterodimerization variants (including skew and/or pI variants)can be optionally and independently combined in any way, as long as theyretain their “strandedness” or “monomer partition.”

In the case of pI variants, while embodiments finding particular use areshown in the figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

In addition, any of the heterodimerization variants, may also beindependently and optionally combined with other variants describedherein including, but not limited to, Fc ablation variants, FcRnvariants, and/or half/life extension variants as generally outlinedherein.

Exemplary combinations of modifications are shown in FIG. 8 and thebackbone sequences in FIG. 9 (homodimeric backbones) and 10(heterodimeric backbones). In certain embodiments, the IL-7-Fc fusionprotein is heterodimeric and includes a combination of Fc domainmodifications as depicted in FIG. 8A. In some embodiments, theheterodimeric IL-7-Fc fusion protein includes a first monomer having afirst Fc domain with the backbone sequence of any one of the “monomer 1”backbones in FIG. 10 and a second Fc domain with the backbone sequenceof a corresponding “monomer 2” backbone in FIG. 10. In some embodiments,the IL-7-Fc fusion protein is homodimeric and includes a combination ofFc domain modifications as depicted in FIG. 8B. In certain embodiments,the homodimeric IL-7-Fc fusion protein includes a first monomer with afirst Fc domain and a second monomer with a second Fc domain, where thefirst and second Fc domains each have the sequence of any of thebackbone sequences in FIG. 9.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S, isosteric pI variantsQ295E/N384D/Q418E/H421D, and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants S364K/E357Q and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first and second monomers each also include aN297A or N297S amino acid substitution that removes glycosylation. Insome embodiments, the first monomer includes a first Fc domain withmodificationsC220S/E233P/L234V/L235A/G236del/S267K/Q295E/L368D/K370S/384D/Q418E/N421Dand optionally M428L/N434S and the second monomer includes a second Fcdomain with modificationsC220S/E233P/L234V/L235A/G236del/S267K/S364K/E357Q and optionallymodifications M428L/N434S, according to the EU index.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S, isosteric pI variantsQ295E/N384D/Q418E/N421D, and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants S364K and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first monomer includes a first Fc domain withmodifications C220S/E233P/L234V/L235A/G236del/S267K/Q295E/L368D/K370S/N384D/Q418E/N421D and optionally M428L/N434S and the second monomerincludes a second Fc domain with modificationsC220S/E233P/L234V/L235A/G236del/S267K/S364K and optionally modificationsM428L/N434S, according to the EU index.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368E/K370S, isosteric pI variantsQ295E/N384D/Q418E/N421D, and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants S364K and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first monomer includes a first Fc domain withmodifications C220S/E233P/L234V/L235A/G236del/S267K/Q295E/L368E/K370S/N384D/Q418E/N421D and optionally M428L/N434S and the second monomerincludes a second Fc domain with modificationsC220S/E233P/L234V/L235A/G236del/S267K/S364K and optionally modificationsM428L/N434S, according to the EU index.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants K360E/Q362E/T411E, isosteric pI variantsQ295E/N384D/Q418E/N421D, and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants D401K and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first monomer includes a first Fc domain withmodificationsC220S/E233P/L234V/L235A/G236del/S267K/Q295E/K360E/Q362E/384D/T411E/Q418E/N421Dand optionally M428L/N434S and the second monomer includes a second Fcdomain with modifications C220S/E233P/L234V/L235A/G236del/D401K andoptionally modifications M428L/N434S, according to the EU index.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S and a variant that ablates Fab armexchange, S228P, and the second monomer includes a second Fc domain withheterodimeric pI variants S364K/E357Q and S228P to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first monomer includes a first Fc domain withmodifications C220S/L368D/K370S and optionally M428L/N434S and thesecond monomer includes a second Fc domain with modificationsC220S/S228P/S364K/E357Q and optionally modifications M428L/N434S,according to the EU index. In exemplary embodiments, the Fc domains arehuman IgG4 Fc domains.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S and isosteric pI variantsQ295E/N384D/Q418E/N421D and the second monomer includes a second Fcdomain with heterodimer skew variants S364K/E357Q, according to the EUindex. In some embodiments, the first and second monomers each alsoinclude M428L/N434S half-life extension variants. In some embodiments,the first and second monomers each also include a C219S hingemodification. In some embodiments, the first monomer includes a first Fcdomain with modifications Q295E/L368D/K370S/384D/Q418E/N421D andoptionally M428L/N434S and the second monomer includes a second Fcdomain with modifications S364K/E357Q and optionally modificationsM428L/N434S, according to the EU index. In exemplary embodiments, the Fcdomains are human IgG2 Fc domains.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S, isosteric pI variantsQ295E/N384D/Q418E/N421D, and FcKO variant S267K and the second monomerincludes a second Fc domain with heterodimer skew variants S364K/E357Qand FcKO variant S267K, according to the EU index. In some embodiments,the first and second monomers each also include M428L/N434S half-lifeextension variants. In some embodiments, the first and second monomerseach also include a C219S hinge modification. In some embodiments, thefirst monomer includes a first Fc domain with modificationsS267K/Q295E/L368D/K370S/384D/Q418E/N421D and optionally M428L/N434S andthe second monomer includes a second Fc domain with modificationsS267K/S364K/E357Q and optionally modifications M428L/N434S, according tothe EU index. In exemplary embodiments, the Fc domains are human IgG2 Fcdomains.

In some embodiments, wherein the IL-7-Fc fusion protein is a monovalent(i.e., only one IL-7), the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants S364K/E357Q, isosteric pIvariants P217R/P228R/N276K, and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first monomer includes a first Fc domain withmodifications C220S/E233P/L234V/L235A/G236del/S267K/L368D/K370S andoptionally M428L/N434S and the second monomer includes a second Fcdomain with modificationsP217R/C220S/P228R/E233P/L234V/L235A/G236del/S267K/N276K/S364K/E357Q andoptionally modifications M428L/N434S, according to the EU index.

In some embodiments, wherein the dimeric IL-7-Fc fusion protein isbivalent (two IL-7s), the first monomer and second monomer each includean Fc domain with FcKO variants E233/P/L234V/L235A/G236del/S267K. Insome embodiments, the first and second monomers each also includeM428L/N434S half-life extension variants. In some embodiments, the firstand second monomers each also include a C220S hinge amino acidsubstitution. In certain embodiments, the first and second Fc domainincludes the amino acid modificationsC220S/E233P/L234V/L235A/G236del/S267K and optionally modifications428L/N434S, according to the EU index.

The variant Fc domains provided herein can also include 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 additional mutations in addition to the enumeratedmutations.

IV. Interleukin 7

The IL-7-Fc fusion proteins provided herein include at least one IL-7.In some embodiments, the IL-7-Fc fusion protein is a monovalent IL-7-Fcfusion protein that includes one IL-7. In other embodiments, the IL-7-Fcfusion protein is a bivalent IL-7-Fc fusion protein that includes twoIL-7s. The IL-7s that can be used with the IL-7-Fc fusion proteinsprovided herein include wildtype IL-7 (see FIGS. 1-3), functionalfragments of such IL-7s and variants that include 1, 2, 3, 4, 5, 6, 7,8, 9, 10 amino acid modifications as compared to wildtype IL-7 (e.g.,wildtype human IL-7).

In some embodiments, the IL-7 is a variant human IL-7 that is at least90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical to human IL-7. Inparticular embodiments, the IL-7 includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 modifications as compared towildtype human IL-7.

In certain embodiments, the IL-7 includes one or more modifications toreduce heterogeneity that may affect IL-7-Fc fusion protein productionand/or activity. In some embodiments, such IL-7 variants include one ormore modifications to remove one or more potential N-glycosylationsites. In some embodiments, an asparagine (N) of a wildtype IL-7 issubstituted with alanine (A), glutamine (Q), or aspartic acid (D).Exemplary residues that may be modified to reduce heterogeneity includeamino acid residues N70, T72, N91, T93 and N116. Particularmodifications to reduce heterogeneity include amino acid substitutionsN70D, N70Q, N70V, T72V, N91D, N91Q, N91A, N116D, N116Q, N116A,N70D/N91D/N116D, N70Q/N91Q/N116Q, N70A/N91A/N116A, and combinationsthereof. Numbering of such IL-7 modifications described herein are basedon the human IL-7 mature form sequence in FIG. 1, wherein the firstamino acid of the sequence (“D”) is amino acid position 1. Providedherein are compositions that include such a variant IL-7 having one ormore amino acid substitutions that reduce heterogeneity that may affectIL-7-Fc fusion protein production and/or activity.

In certain embodiments, the IL-7 includes one or more modifications toreduce binding affinity for IL-7Rα and/or CD132 and thereby, decreaseIL-7 potency. Such modifications are believed to decrease the antigensink for IL-7 and extend the half-life of the subject IL-7-Fc fusionprotein. Residues which may be modified to reduce binding affinity forIL-7Rα and/or CD132 include K10, Q11, S14, V15, L16, V18, S19, Q22, I30,L35, D48, N50, E52, M69, S71, T72, D74, L77, H78, L80, K81, E84, G85,I88, L89, L128, E137, and N143. Particular modifications to reducebinding affinity for IL-7Rα and/or CD132 include amino acidsubstitutions Q11E, Q22E, I30H, L35Q, L35N, D48N, N50D, E52Q, M69S,M69Q, D74N, D74E, K81R, K81E, E84Q, I88T, I88R, L128R, L128Q, E137Q,N143D, D74N/E84Q, D74N/K81R, and D74N/K81E, and combinations thereof.Numbering of such IL-7 modifications described herein are based on thehuman IL-7 mature form sequence in FIG. 1, wherein “D” is amino acidposition 1. Provided herein are compositions that include such a variantIL-7 having one or more amino acid substitutions that decrease IL-7potency.

Exemplary IL-7 variants that can be included in the subject IL-7-Fcfusion proteins include, but are not limited to those in FIGS. 29 and30. In some embodiments, the IL-7-Fc fusion protein includes one or moreof the IL-7 variants in FIGS. 29 and 30. In certain embodiments, theIL-7-Fc fusion protein includes an IL-7 that includes 1, 2, 3, 4, 5, 6,7, 8, 9, 10 additional modifications as compared to an IL-7 variant inFIGS. 29 and 30.

Although the illustrative sequences as depicted in FIG. 29 includesubstitutions of the asparagine (N) at positions 70, 91, and/or 116 withalanine (A), glutamine (Q), or aspartic acid (D), the asparagine atpositions 70, 91, and/or 116 can be substituted with any amino acid toprevent glycosylation. Additionally or alternatively, the threonine atpositions 72 and 93 and the serine at position 118 can be substitutedwith any amino acid other than threonine and serine to preventglycosylation. Additional engineering approaches as known in the art mayalso be used to prevent glycosylation of the IL-7 moiety.

In some embodiments of the bivalent IL-7-Fc fusion protein providedherein, the fusion protein includes two of the same IL-7s (eitherwildtype or IL-7 variants). In other embodiments, the bivalent IL-7-Fcfusion protein includes two different IL-7s (e.g., two different IL-7variants, or one wildtype IL-7 and one variant IL-7).

In one aspect, provided herein are compositions that include one or moreIL-7 variants described herein.

V. Domain Linkers

In some embodiments of the subject IL-7-Fc fusion protein, an IL-7 iscovalently attached to an Fc domain by a linker (e.g., Fc-L-(IL-7)₁,Fc-L-(IL-7)₂, (IL-7)₁-L-Fc and (IL-7)₂-L-Fc). In some embodiments, thelinker is a “domain linker.” While any suitable linker can be used, manyembodiments utilize a glycine-serine polymer, including for example(GS)_(n), (GSGGS)_(n) (“GSGGS” disclosed as SEQ ID NO: 1), (GGGGS)_(n)(“GGGGS” disclosed as SEQ ID NO: 2), and (GGGS)_(n) (“GGGS” disclosed asSEQ ID NO: 3), where n is an integer of at least 0 (and generally from 0to 1 to 2 to 3 to 4 to 5), as well as any peptide sequence that allowsfor recombinant attachment of the two domains with sufficient length andflexibility to allow each domain to retain its biological function. Incertain cases, useful linkers include (GGGGS)₁ (SEQ ID NO: 2) or(GGGGS)₂ (SEQ ID NO: 4). Illustrative domain linkers are depicted inFIG. 7. In some cases, and with attention being paid to “strandedness”,as outlined below, charged domain linkers can be used as discussedherein.

VI. IL-7-Fc Fusion Protein Formats

Useful dimeric IL-7-Fc fusion protein formats are shown in FIGS. 11, 14,17 and 19. IL-7-Fc fusion proteins provided herein include bivalentIL-7-Fc fusion proteins (FIGS. 11 and 17) and monovalent IL-7-Fc fusionproteins (FIGS. 14 and 19).

A. Bivalent IL-7-Fc Fusion Proteins

In some embodiments, the IL-7 fusion is a bivalent IL-7-Fc fusionprotein that includes a) a first monomer that includes a first IL-7covalently attached to a first Fc domain and b) a second monomer thatincludes a second IL-7 covalently attached to second Fc domain.

Any of the IL-7s described herein can be included in the bivalentIL-7-Fc fusion protein. In some embodiments, the first and second IL-7sare wildtype mature human IL-7 (FIG. 1). In certain embodiments, thefirst and second IL-7s are variant IL-7s that include one or moremodifications as depicted in FIGS. 29 and 30. In certain embodiments,each of the first and second IL-7s of the bivalent IL-7-Fc fusionproteins is a variant IL-7 that includes a modification selected fromN70D, T72V, N91D, N116D, N70D/N91D/N116D, Q11E, Q22E, I30H, L35Q, L35N,D48N, N50D, E52Q, M69S, M69Q, D74N, D74E, K81R, K81E, E84Q, I88T, I88R,L128R, L128Q, E137Q, N143D, D74N/E84Q, D74N/K81R, and D74N/K81E orcombinations thereof. In an exemplary embodiment, the first and secondIL-7 are the same. In other embodiments, the first and second IL-7 aredifferent.

Any Fc domain can be included in the bivalent IL-7-Fc fusion protein,including the wildtype and variant Fc domains described herein. In someembodiments, each Fc domain includes a CH2 and CH3. In certainembodiments, the first and second Fc domains include a hinge, CH2 andCH3. In one embodiment, the first and second Fc domains each have theformula, from N-terminus to C-terminus, hinge-CH2-CH3.

In certain embodiments, each Fc domain of the bivalent IL-7-Fc fusionprotein includes FcKO variants E233/P/L234V/L235A/G236del/S267K. In someembodiments, the first and second Fc domains include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondFc domains each include a C220S hinge amino acid substitution. Incertain embodiments, the first and second Fc domains each include theamino acid modifications C220S/E233P/L234V/L235A/G236del/S267K andoptionally modifications M428L/N434S, according to EU numbering. In anexemplary embodiment, the first Fc domain and the second Fc domain arethe same (homodimeric Fc). In other embodiments, the first and second Fcdomains are different (heterodimeric Fc).

FIG. 8B further depicts amino acid modifications that included in thefirst and second monomers of a homodimeric monovalent IL-7-Fc fusionprotein. Exemplary Fc domain “backbone sequences” that find use in thesubject bivalent IL-7-Fc fusion proteins are depicted in FIG. 9 (for usein homodimeric bivalent IL-7-Fc fusion proteins) and FIG. 10 (for use inheterodimeric bivalent IL-7-Fc fusion proteins).

1. N-Terminal Bivalent IL-7-Fc Fusion Proteins ((IL-7)₂-Fc and(IL-7)₂-L-Fc)

In certain embodiments, the IL-7 of each monomer of the bivalent IL-7-Fcfusion protein is covalently attached to the N-terminus of itsrespective Fc domain on the same monomer (FIG. 11). In some embodiments,the N-terminus of each IL-7 is attached to the N-terminus of each of itsrespective Fc domain on the same monomer. In other embodiments, theC-terminus of each IL-7 is attached to the N-terminus of each of itsrespective Fc domain on the same monomer. In some embodiments, the IL-7is directly attached to the N-terminus of the Fc domain ((IL-7)₂-Fc(FIG. 11A). In other embodiments, the IL-7 is attached to the N-terminusof the Fc domain via a linker ((IL-7)₂-L-Fc, FIG. 11B). Exemplarylinkers that can be included are shown in FIG. 7. In particularembodiments, the linker is (GGGGS)4 (SEQ ID NO: 5). In exemplaryembodiments, each of the monomer of the bivalent IL-7-Fc fusion proteinis according to the formula selected from:

1) [N-(IL-7)-C]-[N-Fc domain-C]

2) [C-(IL-7)-N]-[N-Fc domain-C]

3) [N-(IL-7)-C]-[linker]-[N-Fc domain-C]

4) [C-(IL-7)-N]-[linker]-[N-Fc domain-C]

In the formulas above, “IL-7” is any IL-7 provided herein (see, e.g.,wildtype or variant IL-7 depicted in FIGS. 29 and 30), “Fc domain”refers to any Fc domain provided herein (e.g., wildtype or variant Fcdomains provided herein), and “linker” refers to any linker providedherein (see, e.g., FIG. 9). Further, “N” and “C” refer to the N-terminaland C-terminal orientation of each component in each monomer. In someembodiments, the Fc domain has the formula N-hinge-CH2-CH3-C. In certainembodiments, each of the first and second Fc domains have the formulaN-CH2-CH3-C.

Exemplary bivalent N-terminal IL-7 fusion proteins include XENP27088 andXENP27089 in FIGS. 12 and 13, respectively. Exemplary bivalentN-terminal IL-7 fusion proteins that include variant IL-7s includeXENP29754-XENP28782, as depicted in FIG. 31.

2. C-Terminal Bivalent IL-7-Fc Fusion Proteins (Fc-(IL-7)₂ andFc-L-(IL-7)₂)

In certain embodiments, the IL-7 of each monomer of the bivalent IL-7-Fcfusion protein is covalently attached to the C-terminus of itsrespective Fc domain on the same monomer (FIG. 17). In some embodiments,the N-terminus of each IL-7 is attached to the C-terminus of each of itsrespective Fc domain on the same monomer. In other embodiments, theC-terminus of each IL-7 is attached to the C-terminus of each of itsrespective Fc domain on the same monomer. In some embodiments, the IL-7is directly attached to the C-terminus of the Fc domain (Fc-(IL-7)₂,FIG. 17A). In other embodiments, the IL-7 is attached to the C-terminusof the Fc domain via a linker (Fc-L-(IL-7)₂ FIG. 17B). Exemplary linkersthat can be included are shown in FIG. 7. In particular embodiments, thelinker is (GGGGS)4 (SEQ ID NO: 5). In exemplary embodiments, each of themonomer of the bivalent IL-7-Fc fusion protein is according to theformula selected from:

1) [N-Fc domain-C]-[N-(IL-7)-C]

2) [N-Fc domain-C]-[C-(IL-7)-N]

3) [N-Fc domain-C]-[linker]-[N-(IL-7)-C]

4) [N-Fc domain-C]-[linker]-[C-(IL-7)-N]

In the formulas above, “IL-7” is any IL-7 provided herein (see, e.g.,wildtype or variant IL-7 depicted in FIGS. 29 and 30), “Fc domain”refers to any Fc domain provided herein (e.g., wildtype or variant Fcdomains provided herein), and “linker” refers to any linker providedherein (see, e.g., FIG. 7). Further, “N” and “C” refer to the N-terminaland C-terminal orientation of each component in each monomer. In someembodiments, the Fc domain has the formula N-hinge-CH2-CH3-C. In certainembodiments, each of the first and second Fc domains have the formulaN-CH2-CH3-C.

Exemplary bivalent N-terminus IL-7 fusion proteins include XENP27090, asshown in FIG. 18.

B. Monovalent IL-7-Fc Fusion Proteins

In some embodiments, the IL-7 fusion is monovalent IL-7-Fc fusionprotein that includes a) a first monomer that includes a first Fc domainalone (i.e., an “empty Fc”); and b) a second monomer that includes anIL-7 covalently attached to a second Fc domain. See FIGS. 14 and 19.

Any of the IL-7s described herein can be included in the monovalentIL-7-Fc fusion protein. In some embodiments, the IL-7 is wildtype maturehuman IL-7 (FIG. 1). In certain embodiments, the IL-7 is a variant IL-7that includes one or more modifications as depicted in FIGS. 29 and 30.In some embodiments, the IL-7 of the monovalent IL-7-Fc fusion proteinsis a variant IL-7 that includes a modification selected from N70D, T72V,N91D, N116D, N70D/N91D/N116D, Q11E, Q22E, I30H, L35Q, L35N, D48N, N50D,E52Q, M69S, M69Q, D74N, D74E, K81R, K81E, E84Q, I88T, I88R, L128R,L128Q, E137Q, N143D, D74N/E84Q, D74N/K81R, and D74N/K81E or combinationsthereof.

Any Fc domains can be included in the monovalent IL-7-Fc fusion protein,including the wildtype and variant Fc domains described herein. In someembodiments, each Fc domain includes a CH2 and CH3. In certainembodiments, the first and second Fc domains include a hinge, CH2 andCH3. In one embodiment, the first and second Fc domains each have theformula, from N-terminus to C-terminus, hinge-CH2-CH3. In exemplaryembodiments, the first and second Fc domains of the monovalent IL-7-Fcfusion protein are heterodimeric. Modifications for such Fc domains aredescribed in Section III.C above.

In an exemplary embodiments, the monovalent IL-7-Fc fusion protein isheterodimeric. In some heterodimeric embodiments, the first and secondFc domains include the amino acid substitution setL368D/K370S:S364K/E357Q. In some embodiments, the L368D/K370Smodifications are in the first Fc domain and the S364K/E357Qmodifications are in the second domain. In certain heterodimericembodiments, the first Fc domain includes isosteric pI variantsQ295E/N384D/Q418E/H421D.

In certain embodiments, both the first and second Fc domains includeFcKO variants: E233P/L234V/L235A/G236del/S267K, according to the EUnumbering.

In some embodiments, the first monomer includes a first Fc domain withheterodimer skew variants L368D/K370S, isosteric pI variantsQ295E/N384D/Q418E/H421D, and FcKO variantsE233P/L234V/L235A/G236del/S267K and the second monomer includes a secondFc domain with heterodimer skew variants S364K/E357Q and FcKO variantsE233P/L234V/L235A/G236del/S267K, according to the EU index. In someembodiments, the first and second monomers each also include M428L/N434Shalf-life extension variants. In some embodiments, the first and secondmonomers each also include a C220S hinge amino acid substitution. Insome embodiments, the first and second monomers each also include aN297A or N297S amino acid substitution that removes glycosylation. Insome embodiments, the first monomer includes a first Fc domain withmodificationsC220S/E233P/L234V/L235A/G236del/S267K/Q295E/L368D/K370S/384D/Q418EN421Dand optionally M428L/N434S and the second monomer includes a second Fcdomain with modificationsC220S/E233P/L234V/L235A/G236del/S267K/S364K/E357Q and optionallymodifications M428L/N434S, according to the EU numbering.

FIG. 8A further depicts amino acid modifications that included in thefirst and second monomers of a heterodimeric monovalent IL-7-Fc fusionprotein. Additional, exemplary Fc domain “backbone sequences” that finduse in the subject monovalent IL-7-Fc fusion proteins are depicted inFIG. 10.

1. N-Terminal Monovalent IL-7-Fc Fusion Proteins ((IL-7)₁-Fc and(IL-7)₁-L-Fc))

In certain embodiments of the monovalent IL-7-Fc fusion protein, theIL-7 is covalently attached to the N-terminus of the second Fc domain onthe second monomer (FIG. 14). In some embodiments, the N-terminus of theIL-7 is attached to the N-terminus of the second Fc domain. In otherembodiments, the C-terminus of the IL-7 is attached to the N-terminus ofthe second Fc domain. In some embodiments, the IL-7 is directly attachedto the N-terminus of the second Fc domain ((IL-7)₁-Fc, FIG. 14A). Inother embodiments, the IL-7 is attached to the N-terminus of the Fcdomain via a linker ((IL-7)₁-L-Fc, FIG. 14B). Exemplary linkers that canbe included are shown in FIG. 7. In particular embodiments, the linkeris (GGGGS)4 (SEQ ID NO: 5). In exemplary embodiments, the second monomerof the monovalent IL-7-Fc fusion protein is selected from:

1) [N-(IL-7)-C]-[N-Fc domain-C]

2) [C-(IL-7)-N]-[N-Fc domain-C]

3) [N-(IL-7)-C]-[linker]-[N-Fc domain-C]

4) [C-(IL-7)-N]-[linker]-[N-Fc domain-C]

In the formulas above, “IL-7” is any IL-7 provided herein (see, e.g.,wildtype or variant IL-7 depicted in FIGS. 29 and 30), “Fc domain”refers to any Fc domain provided herein (e.g., wildtype or variant Fcdomains provided herein), and “linker” refers to any linker providedherein (see, e.g., FIG. 9). Further, “N” and “C” refer to the N-terminaland C-terminal orientation of each component in the second monomer. Insuch embodiments, the first monomer only includes an Fc domain (i.e., an“empty Fc domain”). In some embodiments, the each of the first andsecond Fc domains have the formula N-hinge-CH2-CH3-C. In certainembodiments, each of the first and second Fc domains have the formulaN-CH2-CH3-C.

Exemplary monovalent N-terminal IL-7 fusion proteins include XENP27079and XENP027080, as shown in FIGS. 15 and 16, respectively. Exemplarymonovalent N-terminal IL-fusion proteins that include a variant IL-7include XENP29187-29202, as shown in FIGS. 32 and 33.

2. C-Terminal Monovalent IL-7-Fc Fusion Proteins (Fc-(IL-7)₁ andFc-L-(IL-7)₁)

In certain embodiments of the monovalent IL-7-Fc fusion protein, theIL-7 is covalently attached to the C-terminus of the second Fc domain onthe second monomer (FIG. 19). In some embodiments, the N-terminus of theIL-7 is attached to the C-terminus of the second Fc domain. In otherembodiments, the C-terminus of the IL-7 is attached to the C-terminus ofthe second Fc domain. In some embodiments, the IL-7 is directly attachedto the C-terminus of the second Fc domain (Fc-(IL-7)₁, FIG. 19A). Inother embodiments, the IL-7 is attached to the N-terminus of the Fcdomain via a linker (Fc-L-(IL-7)₁, FIG. 19B). Exemplary linkers that canbe included are shown in FIG. 7. In particular embodiments, the linkeris (GGGGS)4 (SEQ ID NO: 5). In exemplary embodiments, the second monomerof the monovalent IL-7-Fc fusion protein is selected from:

1) [N-Fc domain-C]-[N-(IL-7)-C]

2) [N-Fc domain-C]-[C-(IL-7)-N]

3) [N-Fc domain-C]-[linker]-[N-(IL-7)-C]

4) [N-Fc domain-C]-[linker]-[C-(IL-7)-N]

In the formulas above, “IL-7” is any IL-7 provided herein (see, e.g.,wildtype or variant IL-7 depicted in FIGS. 29 and 30), “Fc domain”refers to any Fc domain provided herein (e.g., wildtype or variant Fcdomains provided herein), and “linker” refers to any linker providedherein (see, e.g., FIG. 9). Further, “N” and “C” refer to the N-terminaland C-terminal orientation of each component in in the second monomer.In such embodiments, the first monomer only includes an Fc domain (i.e.,an “empty Fc domain”). In some embodiments, the each of the first andsecond Fc domains have the formula N-hinge-CH2-CH3-C. In certainembodiments, each of the first and second Fc domains have the formulaN-CH2-CH3-C.

Exemplary monovalent N-terminal IL-7 fusion proteins include XENP27083,as shown in FIG. 20.

VII. Nucleic Acids

In another aspect, provided herein are nucleic acid compositionsencoding the subject IL-7-Fc fusion proteins and IL-7s (e.g., variantIL-7s) described herein. As will be appreciated by those in the art, thenucleic acid compositions will depend on the format of the fusionprotein. Thus, for example, when the format requires two amino acidsequences (e.g., heterodimeric IL-7-Fc fusions), two nucleic acidsequences can be incorporated into one or more expression vectors forexpression. Similarly, for some formats, only one nucleic acid is needed(homodimeric IL-7-Fc fusions), which can be put into one expressionvectors.

As is known in the art, the nucleic acids encoding the monomercomponents of the IL-7-Fc fusion proteins can be incorporated intoexpression vectors as is known in the art, and depending on the hostcells used to produce the heterodimeric or homodimeric IL-7-Fc fusionproteins. Generally, the nucleic acids are operably linked to any numberof regulatory elements (promoters, origin of replication, selectablemarkers, ribosomal binding sites, inducers, etc.). The expressionvectors can be extra-chromosomal or integrating vectors.

The nucleic acids and/or expression vectors are then transformed intoany number of different types of host cells as is well known in the art,including, but not limited to, mammalian, bacterial, yeast, insectand/or fungal cells, with mammalian cells (e.g. CHO cells) beingpreferred.

In some embodiments, particularly heterodimeric IL-7-Fc fusion proteins,nucleic acids encoding each monomer are each contained within a singleexpression vector, generally under different or the same promotercontrols. In certain embodiments, each of the two nucleic acids arecontained on a different expression vector.

The subject IL-7-Fc fusion protein are made by culturing host cellscomprising the expression vector(s) as is well known in the art. Onceproduced, traditional fusion protein or antibody purification steps aredone, including an ion exchange chromatography step. As discussedherein, having the pIs of the two monomers differ by at least 0.5 canallow separation by ion exchange chromatography or isoelectric focusing,or other methods sensitive to isoelectric point. That is, the inclusionof pI variants that alter the isoelectric point (pI) of each monomer sothat each monomer has a different pI and the resulting heterodimericIL-7-Fc fusion protein also has a distinct pI advantageously facilitatesisoelectric purification of the heterodimer (e.g., anionic exchangechromatography, cationic exchange chromatography). These substitutionsalso aid in the determination and monitoring of any contaminatinghomodimers post-purification (e.g., IEF gels, cIEF, and analytical IEXcolumns).

VIII. Biological and Biochemical Functionality of IL-7 ImmunomodulatoryFc Fusion Proteins

Biological activity of the subject IL-7-Fc fusion proteins and variantIL-7s can be assessed using any IL-7 activity assay known in the art.IL-7 is known to bind to IL-7R, which in turn causes Janus kinases(JAKs) associated with the IL-7R (JAK1 and JAK3) to phosphorylate STAT5protein. STAT5 then translocates into the cell nucleus to regulatefurther downstream processes. Thus, in some embodiments, IL-7 activityis assessed by STAT5 phosphorylation in various lymphocyte populations(e.g., CD4+ T cells, CD8+ T cells, CD56+ NK cells, or Tregs, see Example1c and FIG. 21).

The effects of subject IL-7-Fc fusion protein and variant IL-7s on theproliferation of various lymphocyte populations can be assessed usingany method for lymphocyte proliferation, for example, but not limited toCFSE dilution method, Ki67 intracellular staining of immune effectorcells, and ³H-thymidine incorporation method.

Biological activity of the subject IL-7-Fc fusion proteins can also betested in vivo in an animal model, such as a Graft-versus-Host Disease(GVHD) model conducted in immunodeficient mice with engraftment offoreign immune cells (e.g., human PBMCs) (see Example 1D).

Generally, the subject IL-7-Fc fusion proteins are administered topatients in need thereof (e.g., a patient with a cancer) and efficacy isassessed, in a number of ways as described herein. Thus, while standardassays of efficacy can be run, such as cancer load, size of tumor,evaluation of presence or extent of metastasis, etc., immuno-oncologytreatments can be assessed on the basis of immune status evaluations aswell. This can be done in a number of ways, including both in vitro andin vivo assays.

For example, evaluation of changes in immune status (e.g., presence ofICOS+ CD4+ T cells following ipi treatment) along with traditionalmeasurements such as tumor burden, size, invasiveness, LN involvement,metastasis, etc. can be done. Thus, any or all of the following can beevaluated: the inhibitory effects of PVRIG on CD4⁺ T cell activation orproliferation, CD8⁺ T (CTL) cell activation or proliferation, CD8⁺ Tcell-mediated cytotoxic activity and/or CTL mediated cell depletion, NKcell activity and NK mediated cell depletion, the potentiating effectsof PVRIG on Treg cell differentiation and proliferation and Treg- ormyeloid derived suppressor cell (MDSC)-mediated immunosuppression orimmune tolerance, and/or the effects of PVRIG on proinflammatorycytokine production by immune cells, e.g., IL-2, IFN-γ or TNF-αproduction by T or other immune cells.

In some embodiments, assessment of treatment is done by evaluatingimmune cell proliferation, using for example, CFSE dilution method, Ki67intracellular staining of immune effector cells, and ³H-thymidineincorporation method.

In some embodiments, assessment of treatment is done by evaluating theincrease in gene expression or increased protein levels ofactivation-associated markers, including one or more of: CD25, CD69,CD137, ICOS, PD1, GITR, OX40, and cell degranulation measured by surfaceexpression of CD107A.

In general, gene expression assays are done as is known in the art.

In general, protein expression measurements are also similarly done asis known in the art.

In some embodiments, assessment of treatment is done by assessingcytotoxic activity measured by target cell viability detection viaestimating numerous cell parameters such as enzyme activity (includingprotease activity), cell membrane permeability, cell adherence, ATPproduction, co-enzyme production, and nucleotide uptake activity.Specific examples of these assays include, but are not limited to,Trypan Blue or PI staining, ⁵¹Cr or ³⁵S release method, LDH activity,MTT and/or WST assays, Calcein-AM assay, Luminescent based assay,Annexin V staining, Zombie Aqua™ staining and others.

In some embodiments, assessment of treatment is done by assessing T cellactivity measured by cytokine production, measured eitherintracellularly or in culture supernatant using cytokines including, butnot limited to, IFNγ, TNFα, GM-CSF, IL2, IL6, IL4, IL5, IL10, IL13 usingwell known techniques.

Accordingly, assessment of treatment can be done using assays thatevaluate one or more of the following: (i) increases in immune response,(ii) increases in activation of αβ and/or γδ T cells, (iii) increases incytotoxic T cell activity, (iv) increases in NK and/or NKT cellactivity, (v) alleviation of αβ and/or γδ T-cell suppression, (vi)increases in pro-inflammatory cytokine secretion, (vii) increases inIL-2 secretion; (viii) increases in interferon-γ production, (ix)increases in Th1 response, (x) decreases in Th2 response, (xi) decreasesor eliminates cell number and/or activity of at least one of regulatoryT cells (Tregs).

IX. Treatments

Once made, the subject IL-7-Fc fusion proteins find use in a number ofoncology applications, generally by promoting IL-7 related T cellactivation (e.g., T cells are no longer suppressed) and proliferation.

Accordingly, the subject IL-7-Fc fusion proteins provided find use inthe treatment of these cancers.

A. Fusion Protein Compositions for In Vivo Administration

Formulations of the IL-7-Fc fusion proteins used in accordance with thepresent invention are prepared for storage by mixing a fusion proteinhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (as generally outlined inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, buffers, excipients, or stabilizers are nontoxic to recipientsat the dosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

B. Administrative Modalities

The IL-7-Fc fusion proteins and chemotherapeutic agents are administeredto a subject, in accord with known methods, such as intravenousadministration as a bolus or by continuous infusion over a period oftime.

C. Treatment Modalities

In the methods of treatment provided herein, therapy is used to providea positive therapeutic response with respect to a disease or condition(e.g., a cancer). By “positive therapeutic response” is intended animprovement in the disease or condition, and/or an improvement in thesymptoms associated with the disease or condition. For example, apositive therapeutic response would refer to one or more of thefollowing improvements in the disease: (1) a reduction in the number ofneoplastic cells; (2) an increase in neoplastic cell death; (3)inhibition of neoplastic cell survival; (5) inhibition (i.e., slowing tosome extent, preferably halting) of tumor growth; (6) an increasedpatient survival rate; and (7) some relief from one or more symptomsassociated with the disease or condition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the protein or protein portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the heterodimericproteins used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an heterodimeric proteins used in the present invention is about0.1-100 mg/kg.

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

General and specific scientific techniques are outlined in US Publ. App.No. 2015/0307629, US Publ. App. No. 2014/0288275, U.S. Pat. No.9,605,084 and WO 2014/145806, all of which are expressly incorporated byreference in their entirety and particularly for the techniques outlinedtherein.

Example 1: Engineering IL-7 Fusion Proteins

Cytokines such as IL-7 have short half-life (ranging from 6.5-9.8 hoursfor IL-7). Taking IL-2 as an analogous example, high dose treatment isrequired to achieve a concentration of cytokines at the target (e.g.tumor site) sufficient to induce an immune response. However, based onobservations with IL-2, high dose treatment with IL-7 could potentiallyresult in systemic toxicities. In order to address this, various IL-7-Fcfusion proteins were engineered with the aim to enhance serum half-lifethrough FcRn-mediated recycling.

1A: IL-7 Fusion Protein Formats

We engineered various IL-7-Fc fusion protein formats to investigatewhether the various formats affected either the biological activityand/or production of IL-7-Fc fusions.

1A(a): Bivalent N-Terminal IL-7-Fc Fusion

A first IL-7 fusion category we conceived is the bivalent N-terminusIL-7-Fc fusion (cartoon schematics depicted in FIG. 11).

One such format of this category we engineered as a prototype is the(IL-7)₂-Fc format (cartoon schematic depicted in FIG. 11A) whichcomprises two identical monomers, each monomer comprising an IL-7monomer covalently attached to the N-terminus of a homodimeric Fc chain.An illustrative protein of the (IL-7)₂-Fc format is XENP27088, sequencesfor which are depicted in FIG. 12.

Another format of this category we engineered as a prototype is the(IL-7)₂-L-Fc format (cartoon schematic depicted in FIG. 11B) whichcomprises two identical monomers, each monomer comprising an IL-7monomer covalently attached to the N-terminus of a homodimeric Fc chainvia a domain linker. An illustrative protein of the (IL-7)₂-Fc format isXENP27089, sequences for which are depicted in FIG. 13.

1A(b): Monovalent N-Terminal IL-7-Fc Fusion

Another IL-7 fusion category we conceived is the monovalent N-terminusIL-7-Fc fusion (cartoon schematics depicted in FIG. 14).

One such format of this category we engineered as a prototype is the(IL-7)₁-Fc format (cartoon schematic depicted in FIG. 14A) whichcomprises a first monomer comprising an IL-7 monomer covalently attachedto the N-terminus of a first heterodimeric Fc chain, and a secondmonomer comprising a complementary second heterodimeric Fc chain that is“Fc-only” or “empty-Fc”. An illustrative protein of the (IL-7)₁-Fcformat is XENP27079, sequences for which are depicted in FIG. 15.

Another such format of this category we engineered as a prototype is the(IL-7)₁-L-Fc format (cartoon schematic depicted in FIG. 14B) whichcomprises a first monomer comprising an IL-7 monomer covalently attachedto the N-terminus of a first heterodimeric Fc chain via a domain linker,and a second monomer comprising a complementary second heterodimeric Fcchain that is “Fc-only” or “empty-Fc”. An illustrative protein of the(IL-7)₁-L-Fc format is XENP27080, sequences for which are depicted inFIG. 16.

1A(c): Bivalent C-Terminal IL-7-Fc Fusion

Yet another IL-7 fusion category we conceived is the bivalent C-terminusIL-7-Fc fusion (cartoon schematics depicted in FIG. 17).

One such format of this category we conceived is the Fc-(IL-7)₂ format(cartoon schematic depicted in FIG. 17A) which comprises two identicalmonomers, each monomer comprising an IL-7 monomer covalently attached tothe C-terminus of a homodimeric Fc chain.

Another format of this category we engineered as a prototype is theFc-L-(IL-7)₂ format (cartoon schematic depicted in FIG. 17B) whichcomprises two identical monomers, each monomer comprising an IL-7monomer covalently attached to the C-terminus of a homodimeric Fc chainvia a domain linker. An illustrative protein of the Fc-L-(IL-7)₂-Fcformat is XENP27090, sequences for which are depicted in FIG. 18.

1A(d): Monovalent C-Terminal IL-7-Fc Fusion

Another IL-7 fusion category we conceived is the monovalent C-terminusIL-7-Fc fusion (cartoon schematics depicted in FIG. 19).

One such format of this category we conceived is the Fc-(IL-7)₁ format(cartoon schematic depicted in FIG. 19A) which comprises a first monomercomprising an IL-7 monomer covalently attached to the C-terminus of afirst heterodimeric Fc chain, and a second monomer comprising acomplementary second heterodimeric Fc chain that is “Fc-only” or“empty-Fc”.

Another such format of this category we engineered as a prototype is theFc-L-(IL-7)₁ format (cartoon schematic depicted in FIG. 19B) whichcomprises a first monomer comprising an IL-7 monomer covalently attachedto the C-terminus of a first heterodimeric Fc chain via a domain linker,and a second monomer comprising a complementary second heterodimeric Fcchain that is “Fc-only” or “empty-Fc”. An illustrative protein of theFc-L-(IL-7)₁ format is XENP27083, sequences for which are depicted inFIG. 20.

1B: Production of Prototype IL-7 Fusion Proteins

To produce XENP27088, an illustrative IL-7-Fc fusion of the (IL-7)₂-Fcformat, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding an Fc fusion partner(e.g. homodimeric IgG1 Fc chain as depicted in FIG. 9 as “Homodimeric FcBackbone 1” and as SEQ ID NO: 32).

To produce XENP27089, an illustrative IL-7-Fc fusion of the (IL-7)₂-L-Fcformat, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding a domain linker and anFc fusion partner (e.g. homodimeric IgG1 Fc chain as depicted in FIG. 9as “Homodimeric Fc Backbone 1” and as SEQ ID NO: 32).

To produce XENP27079, an illustrative IL-7-Fc fusion of the (IL-7)₁-Fcformat, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding an Fc fusion partner(e.g. heterodimeric IgG1 Fc chain as depicted in FIG. 10 as“Heterodimeric Fc Backbone monomer 2 and as SEQ ID NO: 39). Anadditional pTT5 expression vector coding for an empty-Fc (e.g. acorresponding second heterodimeric Fc chain as depicted in FIG. 10 as“Heterodimeric Fc Backbone 1 monomer 1” and as SEQ ID NO: 36) was alsoused.

To produce XENP27080, an illustrative IL-7-Fc fusion of the (IL-7)₁-L-Fcformat, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding a domain linker and anFc fusion partner (e.g. heterodimeric IgG1 Fc chain as depicted in FIG.10 as “Heterodimeric Fc Backbone monomer 2 and as SEQ ID NO: 39). Anadditional pTT5 expression vector coding for an empty-Fc (e.g. acorresponding second heterodimeric Fc chain as depicted in FIG. 10 as“Heterodimeric Fc Backbone 1 monomer 1” and as SEQ ID NO: 36) was alsoused.

To produce XENP27090, an illustrative IL-7-Fc fusion of the Fc-L-(IL-7)₂format, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding an Fc fusion partner(e.g. e.g. homodimeric IgG1 Fc chain as depicted in FIG. 9 as“Homodimeric Fc Backbone 1” and as SEQ ID NO: 32) and a domain linker.

To produce XENP27083, an illustrative IL-7-Fc fusion of the Fc-L-(IL-7)₁format, plasmid coding for the IL-7 monomer was constructed by standardgene synthesis, followed by isothermal cloning (Gibson assembly) orsubcloning into a pTT5 expression vector coding an Fc fusion partner(e.g. heterodimeric IgG1 Fc chain as depicted in FIG. 10 as“Heterodimeric Fc Backbone monomer 2 and as SEQ ID NO: 39) and a domainlinker. An additional pTT5 expression vector coding for an empty-Fc(e.g. a corresponding second heterodimeric Fc chain as depicted in FIG.10 as “Heterodimeric Fc Backbone 1 monomer 1” and as SEQ ID NO: 36) wasalso used.

Proteins were produced by transient transfection in HEK293E cells.Bivalent IL-7-Fc fusions (e.g. (IL-7)₂-Fc) were purified using protein Achromatography. Monovalent IL-7-Fc fusions (e.g. (IL-7)₁-Fc) werepurified by a two-step purification process comprising protein Achromatography (purification part 1) followed by ion exchangechromatography (purification part 2).

It should be noted that fusions of the monovalent formats (e.g.(IL-7)₁-Fc) comprise a first heterodimeric Fc region (engineered with ahigher pI; pI(+) e.g. a heterodimeric IgG1 Fc chain as depicted in FIG.10 as “Heterodimeric Fc Backbone monomer 2 and as SEQ ID NO: 39) and acorresponding second heterodimeric Fc region (engineered with a lowerpI; pI(−) e.g. a heterodimeric Fc chain as depicted in FIG. 10 as“Heterodimeric Fc Backbone 1 monomer 1” and as SEQ ID NO: 36). Wereasoned that we could optimize ion exchange purification (purificationpart 2) by engineering the IL-7 monomer on the heterodimeric Fc regionwith the higher pI as this results in a monovalent (IL-7)₁-Fc(pI(−):pI(+)) heterodimer with a calculated pI of 8.13 and an empty-Fc(pI(−):pI(−)) homodimer with a calculated pI of 6.01. On the other hand,engineering the IL-7 on the heterodimeric Fc region with the lowerhigher pI would result in a monovalent (IL-7)₁-Fc (pI(−):pI(+))heterodimer with a calculated pI of 8.13 and an empty-Fc (pI(−):pI(−))homodimer with a calculated pI of 8.66 and subsequently reduced ionexchange separation resolution.

1C: In Vitro Biological Activity of IL-7 Fusions Proteins

Next, we investigated whether the prototype IL-7 fusion proteins werebiologically active. Following cytokine binding to their receptors,Janus kinases (JAKs) associated with the cytokine receptorsphosphorylate STAT proteins which then translocate into the nucleus toregulate further downstream processes. In particular, IL-7 binds to theIL-7 receptor complex and activates JAK1 and JAK3 which phosphorylateSTAT5. Accordingly in a first set of experiments, we used STAT5phosphorylation as an indicator of biological activity of prototypeIL-7-Fc fusion.

Fresh human PBMCs were incubated with indicated concentrations of theindicated test articles for 15 minutes at 37° C. Following incubation,PBMCs were stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605(RPA-T4), and anti-CD8-AF700 (SK1) antibodies for 30-45 minutes at roomtemperature. Cells were washed and incubated with pre-chilled (−20° C.)90% methanol for 20-60 minutes. After methanol incubation, cells werewashed again and stained with anti-CD25-BV421 (M-A251),anti-CD45RA-BV510 (HI100), anti-FoxP3-AF488 (259D), andanti-pSTAT5-AF647 (pY694) to mark various cell populations and STAT5phosphorylation. The data as depicted in FIG. 21 show that each of theprototype IL-7-Fc fusions were active in inducing STAT5 phosphorylationon various lymphocyte populations and that the particular format of theIL-7-Fc fusions did not impact on the potency of STAT5 signaling.Notably, the data show that the IL-7-Fc fusions were more potent thanrecombinant IL-7. Additionally, the data show that CD4⁺ T cells were themost potent responders to recombinant IL-7 and the various IL-7-Fcfusions.

Ki67 is a protein strictly associated with cell proliferation.Accordingly in another set of experiments, we investigated Ki67expression by various lymphocyte populations following incubation withprototype IL-7 fusion proteins as an indicator of cell proliferation.

Fresh human PBMCs were incubated with indicated concentrations of theindicated test articles for 4 days. After incubation, cells were stainedwith anti-CD3-PE (OKT3), anti-CD4-FITC (RPA-T4), anti-CD8-BV510 (SK1),anti-CD16-BV421 (3G8), anti-CD25-PerCP/Cy5.5 (M-A251),anti-CD45RA-APC/Fire750 (HI100), and anti-CD56-BV605 (5.1H11) to gatefor various lymphocyte populations. Staining for intracellular Ki67 wasperformed using anti-Ki-67-PE/Cy7 and Foxp3/Transcription FactorStaining Buffer Set (Thermo Fisher Scientific, Waltham, Mass.).Consistent with the above, the data as depicted in FIG. 22 show thateach of the prototype IL-7-Fc fusions were active in inducingproliferation of various lymphocyte populations, that the particularformat of the IL-7-Fc fusions did not impact on the potency ofproliferative activity, that the IL-7-Fc fusions were more potent thanrecombinant IL-7, and that CD4⁺ T cells were the most potent respondersto recombinant IL-7 and the various IL-7-Fc fusions.

1D: In Vivo Biological Activity of IL-7 Fusions Proteins

The IL-7-Fc fusions were evaluated in a Graft-versus-Host Disease (GVHD)model conducted in NSG (NOD-SCID-gamma) immunodeficient mice. When theNSG mice are engrafted with human PBMCs, the human PBMCs develop anautoimmune response against mouse cells and subsequently GVHD. As such,GVHD is a model for potential anti-tumor response. Treatment ofhuPBMC-engrafted NSG mice with IL-7-Fc fusions should enhancedproliferation of the engrafted T cells and enhance GVHD.

Accordingly in a pilot study, NSG mice were engrafted with 10×10⁶ humanPBMCs via IV-OSP on Day −1 and dosed intraperitoneally with prototypeIL-7-Fc fusion XENP27080 on days 0, 7, and 14. Controls used were PBSand XENP16432 (a bivalent anti-PD-1 mAb, a checkpoint inhibitor whichenhances GVHD by de-repressing the engrafted human T cells; sequencesdepicted in FIG. 23). Body weights were assessed twice per week as anindicator of GVHD (change in body weight as a percentage of initial bodyweight depicted in FIG. 24), and blood was drawn on Days 7, 10, and 14to assess expansion of various lymphocytes (data for which are depictedrespectively in FIGS. 25-27) and cytokine secretion (data for which aredepicted in FIG. 28).

The data show that the IL-7-Fc fusion XENP27080 had significantlyenhanced expansion of CD45⁺, CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells,and NK cells by Day 7 in comparison to PBS control. Further, the datashow that XENP27080 had significantly enhanced expansion of CD45⁺, CD3⁺T cells, CD4⁺ T cells, CD8⁺ T cells, and NK cells by Day 14 incomparison to both PBS control and checkpoint blockade by XENP16432(statistics performed on log-transformed data using unpaired t-test).Furthermore XENP27080 significantly enhanced body weight loss on Days 13and 17 in comparison to checkpoint blockade by XENP16432 (statisticsperformed on data using unpaired t-test), and resulted in death of 2mice by Day 20. Finally, the data show that XENP27080 induced enhancedsecretion of IFNγ, IL-10, and CD25 over the duration of the study.

Example 2: Engineering IL-7 Variants

2A: Engineering IL-7 Variants with Reduced Heterogeneity

Biophysical characterization of the IL-7-Fc fusions indicatedsubstantial heterogeneity which may affect production and/or activity.Accordingly, we sought to engineer IL-7 variants to reduceheterogeneity. In particular, we engineered substitutions to removepotential N-glycosylation sites (N70, N91, and N116) to examine theimpact of glycosylation on protein heterogeneity. Sequences forillustrative variants are depicted in FIG. 29.

Variant (IL-7)₂-L-Fc fusions comprising variant IL-7 as described abovefor removing potential N-glycosylation sites were engineered andproduced as generally described in Example 1B, sequences for which aredepicted in FIG. 31 as XENP28759, XENP28760, XENP28766, XENP28767, andXENP28770. The proteins were analyzed electrophoretically via CEF usingLabChip GXII Touch HT (PerkinElmer, Waltham, Mass.) using ProteinExpress Assay LabChip and Protein Express Assay Reagent Kit carried outusing the manufacturer's instructions. Samples were run undernon-reducing conditions, and gel image is show in FIG. 34. Bands forXENP28759, XENP28760, XENP28766, and XENP28770 were less diffuse thanband for XENP27089 indicating a reduction in heterogeneity.

2B: Engineering IL-7 Variants to Reduce Affinity/Potency (Round 1)

We reasoned that decreasing the affinity of IL-7 for the IL-7 receptorcomplex (and by extension, decreasing their potency) would decreaseantigen sink and extend the half-life of the IL-7 fusion proteins. Weidentified residues K10, Q11, S14, V15, L16, V18, S19, Q22, I30, L35,D48, N50, E52, M69, S71, T72, D74, L77, H78, L80, K81, E84, G85, I88,L89, L128, E137, and N143 as suitable for engineering efforts.Illustrative IL-7 variants engineered with substitutions at some of theabove residues are depicted in FIG. 30. Variant (IL-7)₂-L-Fc fusionscomprising variant IL-7 as described above were engineered and producedas generally described in Example 1B, sequences for which are depictedin FIG. 31.

We used Octet, a BioLayer Interferometry (BLI)-based method, toinvestigate the effect of substitutions described in Example 2B (as wellas Example 2A) on the affinity of IL-7 for IL-7Rα. Experimental stepsfor Octet generally included the following: Immobilization (capture ofligand to a biosensor); Association (dipping of ligand-coated biosensorsinto wells containing serial dilutions of the analyte); and Dissociation(returning of biosensors to well containing buffer) in order todetermine the affinity of the test articles. A reference well containingbuffer alone was also included in the method for background correctionduring data processing. In particular, anti-human Fc (AHC) biosensorswere used to capture each of the IL-7-Fc fusions and dipped intomultiple concentrations of IL-7Rα-His (Sino Biological, Wayne, Pa.).Analysis was performed by global fitting of binding data with a 1:1Langmuir binding model, and the resulting maximum binding responses aredepicted in FIG. 35. The data show that we engineered IL-7-Fc fusionswith a range of binding capacity for IL-7Rα with several variantsdemonstrating drastically reduced binding in comparison to WT IL-7-Fcfusion. Notably, several of the IL-7-Fc fusions comprising IL-7 variantsengineered for reduced heterogeneity also demonstrated reduced binding.

2C: In Vitro Potency of Variant IL-7-Fc Fusions

Next, we engineered IL-7-Fc fusions in the (IL-7)₁-Fc and (IL-7)₁-L-Fcformats incorporating substitutions found in Example 2B to contributethe greatest reduction in maximum IL-7Rα binding response, sequences forwhich are depicted in FIGS. 32-33. We investigated the in vitro activityof the variant IL-7-Fc fusions in a STAT5 phosphorylation assay toconfirm that the reduced binding response contributed to reducedpotency.

In a first experiment, we tested the variant IL-7-Fc fusions in the(IL-7)₁-Fc format. Human PBMCs were stimulated with variousconcentrations of the indicated test articles for 15 minutes at 37° C.PBMCs were then stained with anti-CD3-BUV395 (UCHT1), anti-CD4-BV605(OKT4), anti-CD8-BV421 (SK1), anti-CD45RA-BV785 (H100), andanti-CD45RO-BV510 (UCHL1). Cells were then permeabilized using PerFixEXPOSE (Beckman Coulter, Indianapolis, Ind.). Followingpermeabilization, cells were stained with anti-pSTAT5-Alexa647 (pY694)and analyzed for STAT5 phosphorylation on various lymphocytepopulations, data for which are depicted in FIGS. 36-37. The data showthat (IL-7)₁-Fc fusions comprising D74N and K81E both demonstratedreduced potency compared to WT (albeit, a much greater reduction inpotency by K81E), and combining the two substitutions D74N/K81E provedsynergistic and demonstrated the greatest reduction in potency which isconsistent with the binding data in Example 2C. Furthermore, the datashow that the IL-7-Fc fusions were generally more potent on CD4⁺ T cellscompared to CD8⁺ T cells which is consistent with the data observed inExample 1C (in vitro) and Example 1D (in vivo). Additionally, the datashows that the variant (IL-7)₁-Fc fusions show similar potency ininduction of CD4⁺ memory T cells and CD4⁺ naive T cells, but are morepotent in induction of CD8⁺ naïve T cells than CD8⁺ memory T cells.

Next, we tested the variant IL-7-Fc fusions in the (IL-7)₁-L-Fc formatwhich comprises linkers between the IL-7 monomer and the Fc component.Human PBMCs were stimulated with various concentrations of the indicatedtest articles for 15 minutes at 37° C. PBMCs were then stained withanti-CD3-BUV395 (UCHT1), anti-CD4-BV605 (SK3), anti-CD8-BV421 (SK1),anti-CD45RA-BV785 (H100), and anti-CD45RO-BV510 (UCHL1). Cells were thenpermeabilized using PerFix EXPOSE (Beckman Coulter, Indianapolis, Ind.).Following permeabilization, cells were stained with anti-pSTAT5-Alexa647(pY694) and analyzed for STAT5 phosphorylation on various lymphocytepopulations, data for which are depicted in FIGS. 38-39. Consistent withthe data above for variant (IL-7)₁-Fc fusions, (IL-7)₁-L-Fc fusionscomprising D74N and K81E both demonstrated reduced potency compared toWT, and combining the two substitutions D74N/K81E demonstrated thegreatest reduction in potency. Notably, the data shows that the linkerdoes not impact the potency of the IL-7-Fc fusions.

1. A dimeric Fc fusion protein comprising: (a) a first monomercomprising a first IL-7 and a first Fc domain, wherein said IL-7 iscovalently attached to said first Fc domain; and (b) a second monomercomprising a second IL-7 and a second Fc domain, wherein said secondIL-7 is covalently attached said second Fc domain. 2.-17. (canceled) 18.A heterodimeric Fc fusion protein comprising: a) a first monomerconsisting of first Fc domain; and b) a second monomer comprising anIL-7 and a second Fc domain, wherein said IL-7 is covalently attached tosaid second Fc domain; wherein said first and said second Fc domainscomprise modifications promoting heterodimerization of said first andsaid second Fc domains.
 19. The heterodimeric Fc fusion proteinaccording to claim 18, wherein said IL-7 is attached to the N-terminusof said second Fc domain.
 20. The heterodimeric Fc fusion proteinaccording to claim 18, wherein said IL-7 is attached to the C-terminusof said second Fc domain.
 21. The heterodimeric Fc fusion proteinaccording to claim 18, wherein said first monomer consists of said firstFc domain.
 22. The heterodimeric Fc fusion protein according to claim18, wherein said modifications promoting heterodimerization of saidfirst and second Fc domains are a set of amino acid substitutionsselected from the group consisting of L368D/K370S and S364K; L368D/K370Sand S364K/E357L; L368D/K370S and S364K/E357Q; T411E/K360E/Q362E andD401K; L368E/K370S and S364K; K370S and S364K/E357Q andT366S/L368A/Y407V:T366W (optionally including a bridging disulfide,T366S/L368A/Y407V/Y349C:T366W/S354C), according to EU numbering.
 23. Theheterodimeric Fc fusion protein according to claim 18, wherein said IL-7is attached to said second Fc domain using a domain linker.
 24. Theheterodimeric Fc fusion protein according to claim 23, wherein saiddomain linker is selected from any one of the domain linkers in FIG. 7.25. (canceled)
 26. The heterodimeric Fc fusion protein according toclaim 18, wherein said first and/or said second Fc domains have anadditional set of amino acid substitutions consisting of G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,E233P/L234V/L235A/G236del/S267K, and C219S, C220S, S228P, G236R/L328R,E233P/L234V/L235A/G236del/S239K, E233P/L234V/L235A/G236del/S239K/A327G,E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,E233P/L234V/L235A/G236del/S267K, andC220S/E233P/L234V/L235A/G236del/S267K according to EU numbering.
 27. Theheterodimeric fusion protein according to claim 18, wherein said firstand/or second Fc domains comprise a further amino acid substitutionselected from the group consisting of M428L, N434S, and M428L/N434S,according to EU numbering.
 28. (canceled)
 29. The dimeric fusion proteinaccording to claim 18, wherein the IL-7 is a variant IL-7 comprising oneor more amino acid substitutions, wherein said amino acid substitutionscomprise: N70D, N70Q, N70V, T72V, N91D, N91Q, N91A, N116D, N116Q, N116A,N70D/N91D/N116D, N70Q/N91Q/N116Q, N70A/N91A/N116A, Q11E, Q22E, I30H,L35Q, L35N, D48N, N50D, E52Q, M69S, M69Q, D74N, D74E, K81R, K81E, E84Q,I88T, I88R, L128R, L128Q, E137Q, N143D, D74N/E84Q, D74N/K81R, andD74N/K81E as compared to wild type IL-7.
 30. (canceled)
 31. A nucleicacid composition comprising a first nucleic acid encoding said firstmonomer of claim 18 and a second nucleic acid encoding said secondmonomer of claim
 18. 32. An expression vector composition comprising: afirst expression vector comprising said first nucleic acid and a secondexpression vector comprising said second nucleic acid of claim
 31. 33. Ahost cell comprising said composition of
 32. 34. A method of making aheterodimeric fusion protein comprising culturing the host cell of claim33 and recovering said heterodimeric fusion protein from the cellculture.
 35. A composition comprising a variant human IL-7, said varianthuman IL-7 comprising one or more amino acid substitutions, wherein saidamino acid substitutions comprise: N70D, N70Q, N70V, T72V, N91D, N91Q,N91A, N116D, N116Q, N116A, N70D/N91D/N116D, N70Q/N91Q/N116Q,N70A/N91A/N116A, Q11E, Q22E, 130H, L35Q, L35N, D48N, N50D, E52Q, M69S,M69Q, D74N, D74E, K81R, K81E, E84Q, I88T, I88R, L128R, L128Q, E137Q,N143D, D74N/E84Q, D74N/K81R, and D74N/K81E as compared to wild typehuman IL-7. 36.-56. (canceled)